Method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor

ABSTRACT

One embodiment of the present invention relates to a pharmaceutical composition, which includes a therapeutically effective amount of at least one anti-MIF antibody; and at least one pharmaceutically acceptable carrier. Another embodiment of the present invention relates to a pharmaceutical composition, which includes a therapeutically effective amount at least one anti-CD74 antibody; and at least one pharmaceutically acceptable carrier. Another embodiment of the present invention relates to a pharmaceutical composition, which includes a therapeutically effective amount of at least one anti-TNFR antibody; a therapeutically effective amount of at least one anti-MIF antibody; and at least one pharmaceutically acceptable carrier. Other embodiments of the present invention relate to methods of treating or preventing cardiac dysfunction, cardiodepression, burn injury-associated cardiac dysfunction, improving cardiac function in a subject following acute myocardial infarction, and identifying an MIF inhibitor.

This application is based on U.S. Provisional Application Nos.60/498,659, filed Aug. 29, 2003; 60/547,054, filed Feb. 25, 2004;60/547,056, filed Feb. 25, 2004; 60/547,057, filed Feb. 25, 2004;60/547,059, filed Feb. 25, 2004, and 60/556,440, filed Mar. 26, 2004,the entire contents of each of which are hereby incorporated byreference for all purposes.

This invention was made with Government support under Grant NumberRO1GM58863 awarded by the National Institutes of Health. The Governmentmay have certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention generally relates to pathology and physiology invertebrate species involving cytokines and other cellular signalingmechanisms, and also diagnostic assays involving cytokines and othercellular signaling mechanisms. Other aspects of the invention relate tomacrophage migration inhibitory factor (MIF) as a myocardial depressantfactor and as a mediator of endotoxin-induced cardiac dysfunction invivo. Other aspects of the invention relate to mediating and/orinhibiting the production or activity of MIF, and compounds,compositions, methods of treating and preventing cardiac dysfunction,sepsis, burn injury or other conditions related to burns. Other aspectsof the invention relate to the MIF release from the heart, liver, andspleen and the role of TNF receptor I/II signaling after LPS challenge.Other aspects of the invention relate to TNF receptor I/II signalingindependent release of MIF into the serum. Other aspects of theinvention relate to the expression of CD74 on cardiomyocytes and itsmediation of cardiac dysfunction.

BACKGROUND OF THE TECHNOLOGY

Macrophage migration inhibitory factor (MIF) is a pluripotent,pro-inflammatory cytokine whose mechanisms of action have beenscrutinized over the past four decades. The current understanding in theart relating to MIF includes studies directed to its crystallization asa trimer, its physiologically relevant oligomerization state; itsputative membrane receptor(s); and the physiologic relevance of itsintracellular enzymatic activity as a tautomerase and oxidoreductase.

Many studies have demonstrated that MIF has an important role indiseases as diverse as rheumatoid arthritis (M. Leech, et al.,“Macrophage Migration Inhibitory Factor in Rheumatoid Arthritis:Evidence of Proinflammatory Function and Regulation by Glucocorticoids”,Arthritis Rheum, 42, 1601-1608 (1999), M. Leech, et al., “Involvement ofMacrophage Migration Inhibitory Factor in the Evolution of Rat AdjuvantArthritis”, Arthritis Rheum., 41, 910-917 (1998), A. Mikulowska, et al.,“Macrophage Migration Inhibitory Factor is Involved in the Pathogenesisof Collagen Type 11-Induced Arthritis in Mice”, J. Immunol., 158,5514-5517 (1997)), delayed-type hypersensitivity (J. Bernhagen, et al.,“An Essential Role for Macrophage Migration Inhibitory Factor in theTuberculin Delayed-Type Hypersensitivity Reaction”, J. Exp. Med., 183,277-282 (1996), H. Y. Lan, et al., “De Novo Renal Expression ofMacrophage Migration Inhibitory Factor During the Development of RatCrescentic Glomerulonephritis”, Am. J. Pathol., 149, 1119-1127 (1996),H. Y. Lan, et al., “Macrophage Migration Inhibitory Factor Expression inHuman Renal Allograft Rejection”, Transplantation, 66, 1465-1471 (1998),H. Y. Lan, et al., “TNF-Alpha Up-Regulates Renal MIF Expression in RatCrescentic Glomerulonephritis”, Mol. Med., 3, 136-144 (1997), T.Shimizu, et al., “Increased production of Macrophage MigrationInhibitory Factor by PBMCs of Atopic Dermatitis”, J. Allergy Clin.Immunol., 104, 659-669 (1999)), inflammatory lung disease (S. C.Donnelly, et al., “Regulatory Role for Macrophage Migration InhibitoryFactor in Acute Respiratory Distress Syndrome”, Nat. Med., 3, 320-323(1997), H. Makita, et al., “Effect of Anti-Macrophage MigrationInhibitory Factor Antibody on Lipopolysaccharide-Induced PulmonaryNeutrophil Accumulation”, Am. J. Respir. Crit. Care Med., 158, 573-579(1998), A. G. Rossi, et al., “Human Circulating Eosinophils SecreteMacrophage Migration Inhibitory Factor (MIF). Potential Role in Asthma”,J. Clin. Invest., 101, 2869-2874 (1998)), cancer (J. Chesney, et al.,“An Essential Role for Macrophage Migration Inhibitory Factor (MIF) inAngiogenesis and Growth of a Murine Lymphoma”, Mol. Med., 5, 181-191(1999), M. T. del Vecchio, et al., “Macrophage Migration InhibitoryFactor in Prostatic Adenocarcinoma: Correlation with Tumor Grading andCombination Endocrine Treatment-Related Changes”, Prostate, 45, 51-57(2000), J. D. Hudson, et al., “Al Proinflammatory Cytokine Inhibits p53Tumor Suppressor Activity”, J. Exp. Med., 190, 1375-1382 (1990), A.Kamimura, et al., “Intracellular Distribution of Macrophage MigrationInhibitory Factor Predicts the Prognosis of Patients with Adenocarcinomaof the Lung”, Cancer, 89, 334-341 (2000), K. Meyer-Siegler, et al.,“Increased Stability of Macrophage Migration Inhibitory Factor (MIF) inDU-145 Prostate Cancer Cells”, J. Interferon Cytokine Res., 20, 769-778(2000), T. Shimizu, et al., “High Expression of Macrophage MigrationInhibitory Factor in Human Melanoma Cells and Its Role in Tumor CellGrowth and Angiogenesis”, Biochem. Biophys. Res. Commun., 264, 751-758(1999), Takahashi, et al., “Involvement of Macrophage MigrationInhibitory Factor (MIF) in the Mechanism of Tumor Cell Growth”, Mol.Med., 4, 707-714 (1998)), myocardial infarction (M. Takashashi, et al.,“Elevation of Plasma Levels of Macrophage Migration Inhibitory Factor inPatients with Acute Myocardial Infarction”, Am. J. Cardiol., 89, 248-249(2002), M. Takahashi, et al., “Macrophage Migration Inhibitory Factor asa Redox-Sensitive Cytokine in Cardiac Myocytes”, Cardiovasc Res., 52,438-445 (2001), C. M. Yu, et al., “Elevation of Plasma Level ofMacrophage Migration Inhibitory Factor in Patients with Acute MyocardialInfarction”, Am. J. Cardiol., 88, 774-777 (2001)), and septic shock (J.Berhnagen, et al., “MIF is a Pituitary-Derived Cytokine that PotentiatesLethal Endotoxaemia”, Nature, 365, 756-759 (1993), M. Bozza, et al.,“Targeted Disruption of Migration Inhibitory Factor Gene Reveals ItsCritical Role in Sepsis”, J. Exp. Med. 189, 341-346 (1999), T. Calandra,et al., “MIF as a Glucocorticoid-Induced Modulator of CytokineProduction”, Nature, 377, 68-71 (1995), T. Calandra, et al., “Protectionfrom Septic Shock by Neutralization of Macrophage Migration InhibitoryFactor”, Nat. Med., 6, 164-170 (2000), T. Calandra, et al., “MacrophageMigration Inhibitory Factor is a Critical Mediator of the Activation ofImmune Cells by Exotoxins of Gram-Positive Bacteria”, Proc. Natl. Acad.Sci. USA, 95, 11383-11388 (1998)). There is some evidence thatmonoclonal or polyclonal anti-MIF antibodies may affect the pathology ofsepsis, but their role has not been exhaustively characterized inhumans. However, during septic shock, MIF is increased in the plasma ofanimals and humans, and the blockade of MIF activity by monoclonal orpolyclonal antibodies results in a marked improvement in the survival ofanimals with experimentally induced sepsis (M. Bozza, et al., “TargetedDisruption of Migration Inhibitory Factor Gene Reveals Its Critical Rolein Sepsis”, J. Exp. Med. 189, 341-346 (1999), T. Calandra, et al.,“Protection from Septic Shock by Neutralization of Macrophage MigrationInhibitory Factor”, Nat. Med., 6, 164-170 (2000)).

The blockade of MIF activity has been demonstrated with a number ofinhibitors. Blockade of MIF enzymatic activity has been demonstratedwith diverse chemical compounds as shown in U.S. patent application Ser.No. 10/226,299, filed Aug. 23, 2002, now pending. See also, forinstance, U.S. Pat. No. 6,492,428. Antibodies have also been used toblockade MIF activity as shown in U.S. Pat. No. 6,030,615. MIFexpression can also be inhibited using antisense technology as disclosedin U.S. patent application Ser. No. 08/738,947, filed Oct. 24, 1996, nowpending, or U.S. Pat. No. 6,268,151 which further demonstratespharmaceutical formulations that can be used with all theabove-mentioned MIF inhibitors.

Lipopolysaccharide (LPS) depresses intrinsic myocardial contractilityand is thought to be important in myocardial dysfunction that occurs insepsis and septic shock (A. M. Lefer, “Mechanisms of cardiodepression inendotoxin shock”, Circ Shock Suppl 1:1-8 (1979), J. E. Parrillo, et al.,“A circulating myocardial depressant substance in humans with septicshock. Septic shock patients with a reduced ejection fraction have acirculating factor that depresses in vitro myocartdial cellperformance”, J. Clin. Invest. 76:1539-1553 (1985), J. M. Reilly, etal., “A circulating myocardial depressant substance is associated withcardiac dysfunction and peripheral hypoperfusion (lactic acidemia) inpatients with septic shock”, Chest. 95:1072-1080 (1989)). Manypro-inflammatory cytokines are released after an LPS challenge and havebeen shown to directly mediate the observed cardiac dysfunctionincluding TNF-α, IL-1β, IL-6, IL-18, NO, and macrophage migrationinhibitory factor (MIF) (O. Court, et al., “Clinical review: Myocardialdepression sepsis and septic shock”, Crit. Care 6:500-508 (2002), L. B.Garner, et al., “Macrophage migration inhibitory factor is acaridac-derived myocardial depressant factor”, Am. J. Physiol Heart CircPhysiol, 285:H2500-2509 (2003), S. Krishnagopalan, et al., “Myocardialdysfunction in the patient with sepsis”, Curr. Opin. Crit. Care8:376-388 (2002)). We recently described macrophage migration inhibitoryfactor (MIF) as a cardiac derived myocardial depressant factor in amodel of sublethal endotoxin challenge (endotoxicosis) (L. B. Garner, etal., “Macrophage migration inhibitory factor is a cardiac-derivedmyocardial depressant factor”, Am. J. Physiol Heart Circ. Physiol285:H2500-2509 (2003)). An LPS challenge induced the constitutivepresence of the proinflammatory cytokine MIF to be released maximally by12 hours. The release of MIF in this model paralleled the cardiacdysfunction that MIF was shown to mediate which was delayed after LPSchallenge. Neutralization of MIF by anti-MIF antibodies resulted insignificant protection starting at 8 hours and was completed ablated by48 hours ((L. B. Garner, et al., “Macrophage migration inhibitory factoris a cardiac-derived myocardial depressant factor”, Am. J. Physiol HeartCirc. Physiol 285:H2500-2509 (2003))). MIF is unique among theaforementioned cytokines in its delayed release and ability to blockdownstream mediators.

Investigators have previously reported a temporal discordance betweenthe TNF-α levels in the myocardium and the contractile dysfunction thatoccurred during endotoxemia (X. Meng, et al., “TNF-alpha and myocardialdepression in endoxtoxemic rats: temporal discordance of an obligatoryrelationhship”, Am. J. Physiol 275:R502-508 (1998)). That is, cardiacdysfunction did not occur until TNF-α levels had returned to baselinesuggesting that TNF-α is an important sentinel signal for other cardiacdepressants which more directly conspire to cause dysfunction in sepsis.The significance of these early cytokines is unknown, howevertherapeutic strategies against early mediators of septic shock such asanti-IL-1β and anti-TNF-α modalities have been tested in human trials,no benefits have been observed likely due to their early appearance inthe disease process (C. J. Fisher, et al., “Recombinant human inerleukin1 receptor antagonist in the treatment of patients with sepsis syndrome.Results from a randomized, double-blind, placebo-controlled trial”,Phase III rhIL-Ira Sepsis Syndromve Study Group, Jama, 271:1836-1843(1994), C. J. Fisher, et al., “Initial evaluation of human recombinantinterleukin-1 receptor antagonist in the treatment of sepsis syndrome: arandomized, open-label, placebo-controlled multicenter trial”, TheIL-IRA Sepsis Syndrome Study Group, Crit. Care Med., 22:12-21 (1994), C.Natanson, et al., “Selected treatment strategies for septic shock basedon proposed mechanisms of pathogenesis”, Ann. Intern. Med., 120:771-783(1994), K. Reinhart, et al., “Anti-tumor necrosis factor therapy insepsis:update on clinical trials and lessons learned”, Crit. Care Med.,29:S121-125 (2001)).

Macrophage migration inhibitory factor (MIF) is involved in thepathogenesis of several diseases, including sepsis. MIF opposes theanti-inflammatory effects of glucocorticoids, and also significantlyalters tissue metabolism. Although MIF appears to be ubiquitouslyexpressed, there are currently no publications indicating whether MIF isexpressed in the myocardium in vivo, or whether release of MIF from themyocardium or other tissues during sepsis could adversely affect cardiacperformance.

Cardiac dysfunction during sepsis (O. Court, et al., “Clinical review:Myocardial depression sepsis and septic shock”, Crit. Care, 6:500-508(2002), S. Krishnagopalan, et al., “Macrophage Dysfunction in thePatient with Sepsis”, Curr. Opin. Crit. Care, 8, 376-388 (2002)) isassociated with poor outcome in both humans (P. Ammann, et al.,“Elevation of Troponin I in Sepsis and Septic Shock”, Intensive CareMed., 27, 965-969 (2001), C. N. Sessler, et al., “New Concepts inSepsis”, Curr. Opin. Crit. Care, 8, 465-472 (2002)) and animal models(M. Bozza, et al., “Targeted Disruption of Migration Inhibitory FactorGene Reveals Its Critical Role in Sepsis”, J. Exp. Med., 189, 341-346(1999), T. Calandra, et al., “Protection from Septic Shock byNeutralization of Macrophage Migration Inhibitory Factor”, Nat. Med., 6,164-170 (2000)). It has been previously demonstrated that sepsis or burnassociated cardiac dysfunction is primarily due to circulatingmyocardial depressant factors, including TNF-α (B. P. Giroir, et al.,“Inhibition of Tumor Necrosis Factor Prevents Myocardial DysfunctionDuring Burn Shock”, Am. J. Physiol., 267, H118-H124 (1994), Haudek, etal., “Differential Regulation of Myocardial NF Kappa B Following Acuteor Chronic TNF-Alpha Exposure”, J. Mol. Cell Cardiol., 33, 1263-1271(2001), A. Kumar, et al., “Tumor Necrosis Factor Alpha and Interleukin 1Beta are Responsible for in vitro Myocardial Cell Depression Induced byHuman Septic Shock Serum”, J. Exp. Med., 183, 949-958 (1996)). However,since TNF-α is a sentinel, rapid response cytokine, and is gone from thecirculation days or weeks before the resolution of myocardialdysfunction, there remains a need for finding whether additionalmyocardial depressant proteins might exist.

Studies utilizing live bacteria, either by direct injection of E. colii.p. or by cecal ligation and puncture (CLP), have previouslydemonstrated that MIF plasma and/or peritoneal fluid levels increaseseveral hours post challenge, and that antibodies against MIF protectedthe mice from lethal bacterial peritonitis (T. Calandra, et al.,“Protection from septic shock by neutralization of macrophage migrationinhibitory factor”, Nat. Med., 6, 164-170 (2000)). Moreover, mice wereprotected when the antibodies were given as late as 8 h after the onsetof infection (T. Calandra, et al., “Protection from septic shock byneutralization of macrophage migration inhibitory factor”, Nat. Med., 6,164-170 (2000)).

MIF has a number of properties that make it unique among cytokines. MIFis released preformed from numerous cell types including lymphocytes,macrophages, and the anterior pituitary (J. Bernhagen, et al.,“Regulation of the Immune Response by Macrophage Migration InhibitoryFactor: Biological and Structural Features”, J. Mol. Med., 76, 151-161(1998), T. Calandra, et al., “Macrophage Migration Inhibitory Factor(MIF): A Glucocorticoid Counter-Regulator Within the Immune System”,Crit. Rev. Immunol., 17, 77-88 (1997), S. C. Donnelly, et al.,“Macrophage Migration Inhibitory Factor: A Regulator of GlucocorticoidActivity with a Critical Role in Inflammatory Disease”, Mol. Med. Today,3, 502-507 (1997), R. A. Mitchell, et al., “Tumor Growth-PromotingProperties of Macrophage Migration Inhibitory Factor (MIF)”, Semin.Cancer Biol., 10, 359-366 (2000)). However, the list of sources of MIFcontinues to grow and includes other tissues such as lung, liver,adrenal, spleen, kidney, skin, muscle, thymus, skin, and testes (M.Bacher, et al., “Migration Inhibitory Factor Expression inExperimentally Induced Endotoxemia”, Am. J. Pathol., 150, 235-246(1997), G. Fingerle-Rowson, et al., “Regulation of Macrophage MigrationInhibitory Factor Expression by Glucocorticoids in vivo”, Am. J. Pathol,162, 47-56 (2003)). MIF has at least 2 catalytic activities that aredistinct: tautomerase and oxidoreductase activity. To this end,pharmacological inhibitors of MIF tautomerase activity have beendeveloped for the treatment of MIF-related diseases such as sepsis,acute respiratory distress syndrome (ARDS), asthma, atopic dermatitis,rheumatoid arthritis, nephropathy, and cancer (A. Dios, et al.,“Inhibition of MIF Bioactivity by Rational Design of PharmacologicalInhibitors of MIF Tautomerase Activity”, J. Med. Chem., 45, 2410-2416(2002), M. Orita, et al., “Macrophage Migration Inhibitory Factor andthe Discovery of Tautomerase Inhibitors”, Curr. Pharm. Des., 8,1297-1317 (2002)). These diseases have shown benefit from anti-MIFantibodies.

Several investigations indicate that MIF may exert effects by bothdirect and indirect mechanisms. Previous studies have provided evidencethat MIF promotes the release and pharmacodynamic effects of otherpro-inflammatory cytokines. Macrophages expressing anti-sense MIF cDNA(leading to less endogenous MIF) secrete/express significantly lessTNF-α, IL-6, and NO, while NF-κB activity is decreased in response toLPS (44). Therefore, it appears that MIF may directly interact with theLPS signaling pathway (H. Lue, et al., “Macrophage Migration InhibitoryFactor (MIF): Mechanisms of Action and Role in Disease”, MicrobesInfect., 4, 449-460 (2002)). Moreover, MIF knockout (MIFKO) mice, asdemonstrated in U.S. patent application Ser. No. ______ (Attorney DocketNo. 9551-095-27) filed Dec. 19, 2002, which are resistant to lethaldoses of LPS, have lower circulating plasma levels of TNF-α compared towild-type mice at baseline. Upon LPS challenge, they demonstratediminished circulating TNF-α concentrations, increased nitric oxide (NO)concentrations, and unchanged IL-6 and IL-12 concentrations (M. Bozza,et al., “Targeted Disruption of Migration Inhibitory Factor Gene RevealsIts Critical Role in Sepsis”, J. Exp. Med. 189, 341-346 (1999)). WhileMIF appears to promote pro-inflammatory cytokines, the effects of MIFhave been shown to act in a TNF-α-independent manner. When CLP wasperformed in TNF-α knock out mice, a 60% survival rate was seen in miceadministered anti-MIF antibodies compared to a 0% survival rate inwild-type mice (T. Calandra, et al., “Protection from Septic Shock byNeutralization of Macrophage Migration Inhibitory Factor”, Nat. Med., 6,164-170 (2000)).

In relation to cardiac dysfunction not related to sepsis, elevated serumMIF concentrations have also been described in patients following acutemyocardial infarction (M. Takahashi, et al., “Elevation of Plasma Levelsof Macrophage Migration Inhibitory Factor in Patients with AcuteMyocardial Infarction”, Am. J. Cardiol., 89, 248-249 (2002), M.Takahashi, et al., “Macrophage Migration Inhibitory Factor as aRedox-Sensitive Cytokine in Cardiac Myocytes”, Cardiovasc Res., 52,438-445 (2001), C. M. Yu, et al., “Elevation of Plasma Level ofMacrophage Migration Inhibitory Factor in Patients with Acute MyocardialInfarction”, Am. J. Cardiol., 88, 774-777 (2001)), with heretoforeunknown physiologic relevance. Similarly, cultured cardiac myocytes havebeen noted to release MIF in response to hypoxia and hydrogen peroxide(free radical initiator) but not angiotensin II, endothelin-1, IL-1β, orTNF-α (J. Fukuzawa, et al., “Contribution of Macrophage InhibitoryFactor to Extracellular Signal-Regulated Kinase Activation by OxidativeStress in Cardiomyocytes”, J. Biol. Chem., 277, 24889-24895, M.Takahashi, et al., “Macrophage Migration Inhibitory Factor as aRedox-Sensitive Cytokine in Cardiac Myocytes”, Cardiovasc Res., 52,438-445 (2001)). There are many clinical scenarios which couldpotentially trigger myocardial MIF release, thereby adversely affectingcardiac function. Cardiac dysfunction can be manifest through anyirregular condition in the cardiac myocytes and cardiac tissue. Suchdysfunctions include, but are not limited to, mycarditis, endocarditis,pericarditis, rheumatic heart disease, myocardial infarction, arrythmia,fibrillation, cardiogenic shock, ischemia, hypertrophy, cardiomyopathy,angina, heart murmur or palpitation, heart attack or failure, and any ofthe symptoms or defects associated with congenital heart diseasesgenerally.

Macrophage migration inhibitory factor is a expressed in many organsincluding the heart and has been linked with a delayed cardiacdysfunction in a murine model of endotoxicosis (Garner, et al.,“Macrophage Migration Inhibitory Factor is A Cardiac-Derived MyocardialDepressant Factor”, Am. J. Physiol. Heart Circ. Physiol, 258,H2500-H2509 (2003)).

Burn injury results in cardiac injury and contractile dysfunctioninvolving decreased cardiac output, shock, and left ventricular failure(J. T. Murphy, et al., “Evaluation of Troponin-I as An Indicator ofCardiac Dysfunction After Thermal Injury, 45, 700-704, (1998), E. M.Reynolds, et al., “Left Ventricular Failure Complicating SeverePediatric Burn Injuries”, J. Pediatr. Surg. 30, 264-269; discussion269-270 (1995), W. C. Shoemaker, et al., k″Burn Pathophysiology In Man.I. Sequential Hemodynamic Alterations, J. Surg. Res., 14, 64-73 (1973),R. R. Wolfe, et al., “Review: Acute Versus Chronic Response to BurnInjury”, Circ. Shock, 8, 105-115 (1981)). These contractile deficitshave been reported to appear as early as 2 hours after burn injury (J.W. Horton, et al., “Postburn Cardiac Contractile Function andBiochemical Markers of Postburn Cardiac Injury”, J. Am. Coll. Surg.,181, 289-298 (1995)). Several recent studies have elucidated the earlymolecular events which involve an endotoxin signaling pathway includingthe toll-like receptor 4 (Tlr-4), RAK, and NF-KB in response to gutderived factors (D. L. Carlson, et al., “I Kappa B Overexpression inCardiomyocytes Prevents NF-Kappa B Translocation and ProvidesCardioprotection in Trauma”, Am. J. Physiol. Heart Circ. Physiol, 284,H804-814 (2003), J. T. Sambol, et al., “Burn-Induced Impairment ofCardiac Contractile Function is Due to Gut-Derived Factors Transportedin Mesenteric Lymph”, Shock, 18, 272-276 (2002), J. A. Thomas, et al.,“IRAK Contributes to Burn-Triggered Myocardial Contractile Dysfunction”,Am. J. Physiol. Heart Circ. Physiol., 283, H829-836 (2002), J. A.Thomas, et al., “TLR4 Inactivation and rBPI (21) Block Burn-InducedMyocardial Contractile Dysfunction”, Am. J. Physiol. Heart Circ.Physiol., 283, H1645-1655 (2002)).

While endotoxin signaling through the Tlr-4 receptor represents theinitial pathway of burn injury associated cardiac dysfunction, otherinvestigators have demonstrated that early downstream mediators includeTNF-α, IL-1β, and IL-6 (H. Lue, et al., “Macrophage Migration InhibitoryFactor (MIF): Mechanisms of Action and Role in Disease”, MicrobesInfect., 4, 449-460 (2002)). Experimentally, when TNF-α is blocked, burninjury associated cardiac dysfunction is decreased, emphasizing itsimportance as a key regulator of dysfunction (B. P. Giroir, et al.,“Inhibition of Tumor Necrosis Factor Prevents Myocardial DysfunctionalDuring Burn Shock”, Am. J. Physiol., 267, H118-124 (1994)). Whentherapeutic strategies against early mediators of septic shock such asanti-IL-1β and anti-TNF-α modalities have been tested in human trials,no benefits have been observed likely due to their early appearance inthe disease process (C. J. Fisher Jr., et al., “Recombinant HumanInterleukin 1 Receptor Antagonist in the Treatment of patients withSepsis Syndrome, Results from a Randomized, Double-Blind,Placebo-Controlled Trial”, Phase III rhIL-1ra Sepsis Syndrome StudyGroup, Jama. 271, 1836-1843 (1994), C. J. Fisher, et al., “InitialEvaulation of Human Recombinant Interleukin-1 Receptor Antagonist in theTreatment of Sepsis Syndrome: A Randomized, Open-Label,Placebo-Controlled Multicenter Trial”, The IL-IRA Sepsis Syndrome StudyGroup, Crit. Care Med., 22, 12-21 (1994), C. Natanson, et al., “SelectedTreatment Strategies for Septic Shock Based on Proposed Mechanisms ofPathogenesis”, Ann. Intern. Med., 120, 771-783 (1994), K. Reinhart, etal., “Anti-Tumor Necrosis Factor Thereapy in Sepsis: Update on ClinicalTrails and Lessons Learned”, Crit. Care Med., 29, S121-125 (2001)).

Recently, the cytokine known as macrophage migration inhibitory factor(MIF) has been shown to play a key role in sepsis mortality (H. Lue, etal., “Macrophage Migration Inhibitory Factor (MIF): Mechanisms of Actionand Role in Disease”, Micorbes Infect., 4, 449-460 (2002)). In fact,anti-MIF therapy has been shown to improve survival significantly inlethal models of sepsis (cecal ligation and puncture), even when givenup to 8 hours after the insult (T. Calandra, et al., “Protection FromSeptic Shock Byneutralization of Macrophage Migration InhibitoryFactor”, Nat. Med., 6, 164-170 (2000)). Moreover, MIF has been shown toplay a key role in ARDS (K. N. Lai, et al., “Role For MacrophageMigration Inhibitory Factor in Acute Respiratory Distress Syndrome”, J.Pathol., 199, 496-508 (2003)), a common complication of burn injury (M.Bhatia, et al., “Role of Inflammatory Mediators in the Pathophysiologyof Acute Respiratory Distress Syndrome”, J. Pathol., 202, 145-156(2004)). The purpose of this study was then to identify and characterizeMIF as a useful therapeutic target of burn injury associated cardiacmorbidity using a well defined murine model of burn injury (Garner, etal., “Macrophage Migration Inhibitory Factor is a Cardiac-DerivedMyocardial Depressant Factor”, Am. J. Physiol. Heart Circ. Physiol.,285, H2500-2509 (2003)).

Accordingly, there remains a need for therapies for cardiodepression,cardiodysfunction, burn associated morbidities and cardioprotection inwhich no therapy is currently available.

U.S. Pat. No. 6,030,615 relates to methods and compositions for treatinga disease caused by cytokine-mediated toxicity.

U.S. Pat. No. 6,420,188 relates to methods and compositions forantagonizing MIF activity and methods of treating various diseases basedon this activity.

U.S. Pat. No. 6,599,938 relates to methods and compositions forantagonizing MIF activity and methods of treating various diseases basedon this activity.

U.S. Pat. No. 6,645,493 relates to compositions and methods forinhibiting the release and/or biological activity of MIF.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a pharmaceuticalcomposition effective for at least one selected from the group includingtreating and/or preventing cardiac dysfunction in a subject in needthereof, treating and/or preventing irregularity in myocardial activityin a subject in need thereof, treating and/or preventing depression inmyocardial activity in a subject in need thereof, treating and/orpreventing burn-injury associated cardiac dysfunction in a subject inneed thereof, treating and/or preventing cardiac dysfunction followingacute myocardial infarction in a subject in need thereof, treatingand/or preventing cardiodepression in a subject in need thereof, and acombination thereof, which includes:

-   -   an effective amount of at least one anti-MIF antibody; and    -   at least one pharmaceutically acceptable carrier.

Another embodiment of the present invention relates to a pharmaceuticalcomposition effective for at least one selected from the group includingtreating and/or preventing cardiac dysfunction in a subject in needthereof, treating and/or preventing irregularity in myocardial activityin a subject in need thereof, treating and/or preventing depression inmyocardial activity in a subject in need thereof, treating and/orpreventing burn-injury associated cardiac dysfunction in a subject inneed thereof, treating and/or preventing cardiac dysfunction followingacute myocardial infarction in a subject in need thereof, treatingand/or preventing cardiodepression in a subject in need thereof, and acombination thereof, which includes:

-   -   an effective amount of at least one anti-CD74 antibody, and    -   an effective amount of at least one pharmaceutically acceptable        carrier.

Another embodiment of the present invention relates to a pharmaceuticalcomposition effective for at least one selected from the group includingtreating and/or preventing cardiac dysfunction in a subject in needthereof, treating and/or preventing irregularity in myocardial activityin a subject in need thereof, treating and/or preventing depression inmyocardial activity in a subject in need thereof, treating and/orpreventing burn-injury associated cardiac dysfunction in a subject inneed thereof, treating and/or preventing cardiac dysfunction followingacute myocardial infarction in a subject in need thereof, treatingand/or preventing cardiodepression in a subject in need thereof, and acombination thereof, which includes:

-   -   an effective amount of at least one anti-TNFR antibody;    -   an effective amount of at least one anti-MIF antibody; and    -   at least one pharmaceutically acceptable carrier.

Another embodiment of the present invention relates to a method fortreating and/or preventing cardiac dysfunction in a subject, the methodincluding:

-   -   administering to the subject an effective amount of at least one        anti-MIF antibody.

Another embodiment of the present invention relates to a method fortreating and/or preventing burn injury-associated cardiac dysfunction ina subject, the method including:

-   -   administering to the subject an effective amount of at least one        anti-MIF antibody.

Another embodiment of the present invention relates to a method fortreating and/or preventing cardiac dysfunction in a subject, the methodincluding:

-   -   administering to the subject an effective amount of at least one        anti-CD74 antibody.

Another embodiment of the present invention relates to a method forimproving cardiac function in a subject following acute myocardialinfarction, the method including:

-   -   administering to the subject an effective amount of at least one        anti-MIF antibody.

Another embodiment of the present invention relates to a method foridentifying an MIF inhibitor, the method including:

-   -   exposing at least one myocyte to at least one MIF;    -   determining at least one MIF-related myocyte activity;    -   exposing the myocyte to said MIF and at least one candidate        agent;    -   determining the MIF-related myocyte activity in the presence of        the candidate agent; and    -   determining whether the candidate agent affects the MIF-related        myocyte activity.

Another embodiment of the present invention relates to a method fortreating and/or preventing cardiac dysfunction in a subject followingacute myocardial infarction, the method including:

-   -   administering to the subject an effective amount of at least one        anti-TNFR antibody and an effective amount of at least one        anti-MIF antibody.

Another embodiment of the present invention relates to a method fortreating and/or preventing cardiac dysfunction in a subject, the methodincluding:

-   -   administering to the subject an effective amount of at least one        anti-MIF antibody.

Another embodiment of the present invention relates to a method fortreating and/or preventing burn injury-associated cardiac dysfunction ina subject, the method including:

-   -   administering to the subject an effective amount of a        composition which includes at least one anti-MIF antibody and at        least one pharmaceutically acceptable carrier.

Another embodiment of the present invention relates to a method fortreating and/or preventing cardiac dysfunction in a subject, the methodincluding:

-   -   administering to the subject an effective amount of a        composition which includes at least one anti-CD74 antibody and        at least one pharmaceutically acceptable carrier.

Another embodiment of the present invention relates to a method forimproving cardiac function in a subject following acute myocardialinfarction, the method including:

-   -   administering to the subject an effective amount of a        composition which includes at least one anti-MIF antibody and at        least one pharmaceutically acceptable carrier.

Another embodiment of the present invention relates to a method fortreating and/or preventing cardiac dysfunction in a subject followingacute myocardial infarction, the method including:

-   -   administering to the subject an effective amount of a        composition which includes at least one anti-TNFR antibody, at        least one anti-MIF antibody, and at least one pharmaceutically        acceptable carrier.

Another embodiment of the invention relates to a method for at least oneselected from the group including treating and/or preventing cardiacdysfunction in a subject in need thereof, treating and/or preventingirregularity in myocardial activity in a subject in need thereof,treating and/or preventing depression in myocardial activity in asubject in need thereof, treating and/or preventing burn-injuryassociated cardiac dysfunction in a subject in need thereof, treatingand/or preventing cardiac dysfunction following acute myocardialinfarction in a subject in need thereof, treating and/or preventingcardiodepression in a subject in need thereof, and a combinationthereof, which includes administering to said subject an effectiveamount of at least one selected from the group including a smallmolecule MIF inhibitor, salt thereof, prodrug thereof, and a combinationthereof.

Another embodiment of the invention relates to a method for at least oneselected from the group including treating or preventing cardiacdysfunction in a subject in need thereof, treating or preventingirregularity in myocardial activity in a subject in need thereof,treating or preventing depression in myocardial activity in a subject inneed thereof, treating or preventing burn-injury associated cardiacdysfunction in a subject in need thereof, treating or preventing cardiacdysfunction following acute myocardial infarction in a subject in needthereof, treating or preventing cardiodepression in a subject in needthereof, and a combination thereof, which includes administering to asubject in need thereof an effective amount of at least one anti-TNFRantibody; and

-   -   optionally, at least one pharmaceutically acceptable carrier.

DESCRIPTION OF THE FIGURES

The foregoing description will be better understood when read inconjunction with the appended drawings. For the purpose of illustratingthe invention, there is shown in the drawings an embodiment which ispresently preferred, it being understood, however, that this inventionis not limited to the precise arrangements and instrumentalities shown.

FIG. 1: MIF protein release is detected within 12 h of LPS challenge incardiac tissue. Each data point is the mean (+/− standard error) of 3independent Western blot experiments. A representative Western blot isshown below the graph. *p<0.05

FIG. 2: LPS challenge does not upregulate MIF mRNA in cardiac tissue.Each data point in the graph is the mean (+/− standard error) of 3independent Northern blot experiments. A representative Northern blot isshown below the graph. No significant differences between time pointswere identified (p>0.05).

FIG. 3: The presence of MIF in the heart, liver, and spleen before andafter LPS challenge. Preformed MIF in the heart, liver, and spleen (A,D, G) decreases 12 h after LPS challenge (B, E, and H) and isreplenished after 24 h (C, F, and 1) as demonstrated byimmunohistochemistry. Magnification: 100× (Kidney, Spleen), 400×(Heart).

FIG. 4: Cardiac function determined by Langendorff preparation post-rMIFchallenge in C57BL/6J mice and endotoxin-resistant C3H/HeJ micedemonstrates rMIF mediates cardiac dysfunction in an LPS-independentmechanism. Data represents the average of 7 (C3H/HeJ) to 10 (C57BL/6J)independent Langendorff experiments per group. *p<0.05.

FIG. 5: Echocardiographic assessment of the effects of LPS and LPS plusanti-MIF antibody administration on cardiac function. RepresentativeM-mode echocardiograms in wild-type mice at baseline and 8 h after LPSadministration, A and B, respectively. C and D show, respectively,representative echocardiograms in LPS plus anti-MIF treated mice at 8and 48 h. A significant protection in cardiac function (FS %) isobserved in LPS challenged mice when anti-MIF anti-bodies are givenpre-treatment (E). Data represents the average of 9 cardiac cycles from3 mice monitored at multiple time points. *p<0.05.

FIG. 6: Burn model demonstrating inhibition of MIF with anti-MIFantibody following LPS Challenge. The burn data demonstrates inhibitionof MIF with the anti-MIF antibody and restores cardiac functionfollowing burning.

FIG. 7: A graphical representation demonstrating MIF release rate fromthe heart following thermal trauma. Macrophage migration inhibitoryfactor (MIF) is constitutively expressed in cardiac tissue and releasedmaximally 8 hours post-burn injury. Each data point represents the meandensity in arbitrary units (A.U.)/mm²±SE of 3 independent Western blotexperiments. A representative Western blot is shown below the graph. AOne Way ANOVA and a multiple comparison procedure using the Tukey methodwere employed to determine statistical significance compared to thecontrol group (*p<0.05).

FIG. 8: Immunochemistry staining slides demonstrating MIF presence invarious tissue samples. Insert G-MIF, constitutively present in theheart, liver, spleen, lung, and kidney is decreased after burn injury.Preformed MIF in the heart, kidney, and spleen (A, E, 1) decreases 8hours after burn injury (B, F, J), except for liver (M,N) and increasesat 24 hours in heart and kidney (C, G), but not heart or liver (K,O) asdemonstrated by immunohistochemistry. A negative control (secondaryantibody without the primary anti-MIF antibody) consistentlydemonstrates that no background staining is present at each time pointin each organ investigated as represented in the far right column (D, H,L, P). Magnification: 100× (Kidney, Spleen, Liver), 400× (Heart).

FIG. 9: Graphical representations of concentration change of threedifferent cytokines over time following thermal trauma. FIG. 9-Serumconcentrations of MIF (ng/ml), IL-12 (pg/ml), and IL-6 (pg/ml) followingburn injury (A-C, respectively). Data are expressed as the mean±SE ofsix C57BL/6J mice as determined by ELISA and were statistically analyzedusing a One Way-ANOVA with a multiple comparison procedure employing theBonferroni method to determine significance between groups (*p<0.05compared to baseline).

FIG. 10: Graphical representation and representative Northern Blotshowing MIF mRNA upregulation following thermal trauma. Burn injuryupregulates MIF mRNA expression in cardiac tissue significantly by 8hours. MIF and β-actin mRNAs were detected using ³²P radiolabeled probescomplementary to MIF and β-actin mRNAs. Each data point represents themean density in arbitrary units (A.U.)/mm²±SE of 3 independent Northernblot experiments. A representative Northern blot is shown below thegraph. Normalized MIF was determined by multiplying the MIF density bythe relative β-actin density present. A One Way-ANOVA and a multiplecomparison procedure using the Tukey method were employed to determinestatistical significance compared to baseline (*p<0.05).

FIG. 11: Graphical representations of Coronary Flow Rates calculatedthree different ways comparing control measure of flow rate withuntreated thermal trauma versus thermal trauma treated with Anti-MIF.Cardiac function determination by Langendorff preparation 18 hours afterburn injury as a function of coronary flow (A) and Ca²⁺. Cardiacfunction is expressed as the mean±SE of 25 independent Langendorffexperiments (11 sham, 9 burn injury, 5 burn injury after anti-MIFtreatment). Separate analyses were performed for each LVP, +dP/dt_(max),and −dP/dt_(max) as a function of treatment group and coronary flow rateusing a Repeated Measures ANOVA and a multiple comparison procedureemploying the Bonferroni method to determine significant differencesbetween groups (*p<0.05).

FIG. 12: Echocardiographic and graphical representation of the effectsof anti-MIF antibody therapy after burn injury demonstrating thecardioprotective effects of MIF blockade. Echocardiographic assessmentof the effects of anti-MIF antibody therapy after burn injurydemonstrates the cardioprotective effects of MIF blockade.Representative M-mode echocardiograms in wild-type mice at baseline and8 hours after burn injury, A and B, respectively. C and D depictrepresentative echocardiograms in burn injury plus anti-MIF treated miceat 4 and 48 hours, respectively. A significant recovery of cardiacfunction (FS %) is observed in burn injury mice given anti-MIFanti-bodies pre-burn injury (E). Data from each group represent themean±SE of 9 cardiac cycles from 3 mice monitored at multiple timepoints. Cardiac function determined by echocardiography is expressed asfractional shortening % (LVED−LVES/LVED×100)±SE and was analyzed using aOne Way Repeated Measures-ANOVA and a multiple comparison procedureemploying the Tukey Test to determine significant differences betweenspecific groups.

FIG. 13: Serum MIF concentration (fold increase from baseline) followinga 4 mg/kg endotoxin challenge in: (A) wild type mice, (B) TNFR−/− mice,and (C) wild type mice pre-treated (60 minutes) with Enbrel®. Data areexpressed a fraction of the baseline levels of MIF (mean+/− standarderror) of: (A) 6 C57BL/6J mice, (B) 6 TNFR−/− mice, and (C) 3 C57BL/6J(C) pre-treated with Enbrel®. Serum levels were determined by ELISA andwere statistically analyzed using a One Way-ANOVA with a multiplecomparison procedure employing the Bonferroni method to determinesignificance between groups (*p<0.05 compared to baseline).

FIG. 14: MIF protein release is not detected after LPS challenge incardiac tissue. Each data point is the mean (+/− standard error) of 3independent Western blot experiments. A representative Western blot isshown below the graph. *p<0.05

FIG. 15: The presence of MIF in the heart, liver, and spleen before andafter LPS challenge in TNFR−/− mice. Preformed MIF in the heart, liver,and spleen (A, D, G) does not decrease 12 hours after LPS challenge (B,F, J) which is seen in wild type mice as demonstrated byimmunohistochemistry. Magnification: 100× (Kidney, Spleen), 400×(Heart).

FIG. 16: LPS challenge does not upregulate MIF mRNA in cardiac tissue inTNFR−/− mice. Each data point in the graph is the mean (+/− standarderror) of 3 independent Northern blot experiments. A representativeNorthern blot is shown below the graph. No significant differencesbetween time points were identified (p>0.05).

FIG. 17: Cardiac function determined by Langendorff preparationpost-rMIF challenge in C57BL/6J, B6/129S, and TNFR−/− mice demonstratesrMIF mediates cardiac dysfunction independent of TNF-α signaling exvivo. Data represents the average of 6 (C57BL/6J), 4 (B6/129S), and 4(TNFR−/−) independent Langendorff experiments per group. *p<0.05.

FIG. 18: Echocardiographic assessment of the effects of LPS and LPS plusanti-MIF antibody administration on cardiac function in TNFR−/− mice.Representative M-mode echocardiograms in wild-type mice at baseline and4 h after LPS administration, A and B, respectively. C and D show,respectively, representative echocardiograms in LPS plus anti-MIFtreated mice at 4 and 48 hours. A significant protection in cardiacfunction (FS %) is observed in LPS challenged mice when anti-MIFanti-bodies are given pre-treatment (E). Data represents the average of9 cardiac cycles from 3 mice monitored at multiple time points. *p<0.05.

FIG. 19: Serum cytokine determination in wild type and TNFR−/− miceafter LPS challenge. An inflammatory cytokine panel was assayed on theLuminex platform for: (A) TNF-α, (B) IL-1β, (C) IFN-γ, (D) IL-12, (E)IL-10, =(F) IL-6, (G) GM-CSF, and IL-2, IL-4, IL-5 (data not shown).Data are expressed as the mean+/− standard error of serum cytokineconcentrations from 3 independent experimental mice at each time point.

FIG. 20: Serum cytokine determination in wild type after LPS challengewith or without anti-MIF pre-treatment (90 minutes). Shown are themodulated cytokines: (A) IFN-γ and (B) IL-10 as assayed on the Luminexplatform. Data are expressed as the mean+/− standard error of serumcytokine concentrations from 3 independent experimental mice at eachtime point.

FIG. 21: Compares cardiac function (fractional shortening) in post LADligation with LAD only and anti-MIF+LAD.

FIG. 22: Shows the effect of anti-MIF therapy pre-LAD with LAD only andanti-MIF+LAD.

FIG. 23: Presents cardiac function data 48 hours post-LAD for severaltreatment groups.

FIG. 24: Shows the serum troponin concentration 48 hrs post-LAD withpre- and delayed anti-MIF treatment.

FIG. 25: Shows the serum troponin I and MIF concentrations through twoweeks post ligation.

FIG. 26: Shows the organ CD74 constitutive expression and heart CD74post-LPS challenge expression.

FIG. 27: Shows the echocardiography/fractional shortening of severalgroups from t=0 to t=48 hrs post LPS challenge.

FIG. 28: Shows post LPS challenge serum sCD74 release.

FIG. 29: Shows the CD74 series gel. See also Table 5.

FIG. 30: Shows the coronary flow rate v. LVP, +dP/dt max, and −dP/dt maxin CD74 KO Mouse.

FIG. 31. Shows the hours post ligation v. fractional shortening forcontrol, small molecule MIF inhibitor+DMSO/MI and DMSO/MI.

DETAILED DESCRIPTION OF THE INVENTION

Various other objects, features, and attendant advantages of the presentinvention will be more fully appreciated as the same becomes betterunderstood from the following detailed description of the invention,which is not intended to be limiting unless otherwise indicated.

One preferred embodiment of the invention relates to a method oftreatment and bioassay involving macrophage migration inhibitory factor(MIF) as cardiac-derived myocardial depressant factor.

One preferred embodiment of the invention relates to a method oftreatment and/or prevention of cardiac dysfunction associated with burninjury.

One preferred embodiment of the invention relates to the modulation ofMIF as therapy for myocardial infarction.

One preferred embodiment of the invention relates to the modulation ofTNF-α in cardiac dysfunction.

One preferred embodiment of the invention relates to a method ofcardioprotection by inhibition of CD74.

One preferred embodiment of the invention relates to the modulation ofMIF with anti-MIF antibodies.

One preferred embodiment of the invention relates to a bioassay foridentifying agents that inhibit MIF activity.

One preferred embodiment of the invention relates to the inhibition ofMIF with anti-MIF antibody and concomitant restoration of (post-burning)cardiac function.

One preferred embodiment of the invention relates to a method for theimprovement of burn-injury-associated cardiac depression by theadministration of anti-MIF antibodies.

One preferred embodiment of the invention relates to the improvement ofcardiac function following acute myocardial infarction by theadministration of anti-MIF antibody.

One preferred embodiment of the invention relates to the observationthat MIF release from the heart, liver and spleen is dependent uponTNF-α receptor I/II signaling, and thus TNF-α may be a therapeutictarget.

One preferred embodiment of the invention relates to the neutralizationof TNF-α with recombinant human TNFR:Fc.

One preferred embodiment of the invention relates to the neutralizationof MIF activity with one or more anti-MIF antibodies.

One preferred embodiment of the invention relates to the discovery ofTNF-α as an upstream mediator of MIF.

One preferred embodiment of the invention relates to the observationthat CD74 (an MIF receptor) is expressed on cardiomyocytes and is a“critical” mediator of cardiac dysfunction.

One preferred embodiment of the invention relates to the inhibition ofMIF and improving cardioprotection by inhibiting the CD74 receptor withone or more anti-CD74 monoclonal neutralizing antibodies.

One preferred object of the invention relates to a method for treatingand/or preventing cardiac dysfunction, such as an irregularity ordepression in myocardial activity. The method preferably includesadministering an effective amount of a composition comprising amacrophage migration inhibitory factor (MIF) inhibitor. The inhibitorcan be an antibody. The inhibitor can affect a particular MIF activityincluding an enzymatic activity, such as tautomerase activity oroxidoreductase activity.

One preferred embodiment of the invention relates to an assay foridentifying agents that inhibit MIF activity. The assay preferablyinvolves both a myocyte, either in vitro or in vivo, and MIF in thepresence and in the absence of an agent that may inhibit MIF activity.The assay analyzes myocyte activity, for example, using such tools asimmunochemistry or echocardiography, based on the presence of MIF and apotential inhibitor.

One preferred embodiment of the invention relates to a method of usingthis assay to identify an agent that inhibits MIF activity comprisingplacing a myocyte and MIF in the presence of an agent that may inhibitMIF activity, and determining the effect on myocyte activity. Themyocyte may be in vitro or in vivo and the effect may be measuredutilizing immunochemistry or echocardiography.

The present inventors have found that MIF is an inducer of myocardialdysfunction, which is known to contribute significantly to the morbidityand mortality of sepsis in humans. In both human patients and animalmodels, sepsis associated cardiac dysfunction is characterized bybiventricular dilatation, decreased systolic contractility, anddiminished diastolic relaxation. While not wishing to be bound bytheory, it is believed that its pathogenesis is multifactorial, withsystemic and myocardial derived cytokines such as tumor necrosisfactor-alpha (TNF-α) involved in inducing its onset.

One embodiment of the present application is directed to identifyingwhether MIF or other cardiac derived proteins mediate, by paracrine orautocrine mechanisms, myocardial dysfunction in sepsis and other cardiacdiseases. Screening microarray analysis of cardiac gene expression inmice suggests that MIF is expressed in the heart, and is differentiallyregulated after lipopolysaccharide (LPS)-challenge. Given the data thatMIF inhibition improves outcome in animals with experimental sepsis, thefollowing examples were constructed to verify whether MIF was expressedby cardiomyocytes in vivo, whether this expression was altered byendotoxin challenge and that MIF had a physiologically important effecton cardiac function. Several of the examples herein demonstrate cardiacMIF expression in vivo, and determine that MIF depresses cardiacfunction in a sublethal endotoxin challenge in vivo.

MIF is constitutively expressed in the normal myocardium, and isreleased by cardiomyocytes following endotoxin challenge, with cardiactissue levels reaching a nadir 12 hours after challenge. Evidencesupporting a delayed release is seen in the present application bywestern blot and immunohistochemistry demonstrating significant releaseat 12 h from cardiac and spleen tissue, and supported indirectly by thedelayed onset of cardiac protection beginning at eight hours, andcontinuing thereafter. Treatment of LPS challenged mice with anti-MIFmonoclonal antibodies significantly improves in vivo cardiac function asevidenced by improvement in left ventricular shortening fraction.

To further demonstrate MIF's myocardial depressant effects, isolatedbeating mouse hearts (Langendorff perfusion) were perfused with asolution containing a concentration of recombinant MIF (rMIF). Perfusionwith rMIF results in significant depression of both systolic anddiastolic performance. The present inventors have demonstrated that MIFis synthesized by cardiomyocytes in vivo, and released following LPSchallenge. Thus MIF directly mediates cardiac dysfunction and MIF isdemonstrated as a pharmacologic target for the improvement of cardiacfunction in sepsis and other cardiac diseases.

Microarray data on cardiac gene expression highlights that MIF is alsoexpressed in cardiac tissue. Several examples herein indicate that MIFperfusion directly depresses cardiac function in vitro; and moreover,treatment with either of two independent monoclonal antibodies directedagainst MIF mitigates late myocardial depression.

Another preferred embodiment of the invention relates to a method fortreating and/or preventing burn injury associated conditions, includingbut not limited to cardiac dysfunction, such as an irregularity ordepression in myocardial activity. The method preferably includesadministering an effective amount of a composition comprising amacrophage migration inhibitory factor (MIF) inhibitor. The inhibitorcan inhibit MIF activity and/or MIF production. The inhibitor ispreferably an antibody or protein. The inhibitor can affect a particularMIF activity including an enzymatic activity, such as tautomeraseactivity or oxidoreductase activity. The inhibitor can inhibit or blockMIF activity or MIF production in myocardial tissue. The inhibitor canalso inhibit or prevent MIF release, such as inhibitors of the ABCtransporter.

Another preferred embodiment of the invention relates to an assay foridentifying agents that inhibit MIF activity or production. The assaywould involve a myocyte, either in vitro or in vivo, and possibly MIF inthe presence and in the absence of an agent that inhibits MIF activityor MIF production. The assay would analyze myocyte activity, using suchtools as immunochemistry or echocardiography, based on the presence ofMIF and a potential inhibitor.

One embodiment of the invention utilizes the role of MIF in burnassociated cardiac dysfunction in methods of prevention/treatment anddiagnostic assays. Using a murine burn injury model (40% TBSA), thepresent inventors identified that constitutive cardiac MIF significantlydecreased (2.1 fold) 8 hours after burn injury as determined by westernblot analysis. Serum MIF was maximal at 4 hours after burn injury (2.2fold increase). These patterns are consistent with MIF release frompre-formed cytoplasmic stores of cardiac and systemic origin followingburn injury. As seen in the examples herein, to determine the effect ofMIF in cardiac dysfunction seen after burn injury, mice were pre-treatedwith anti-MIF neutralizing monoclonal antibodies. Beginning at 4 hoursafter burn injury (and continuing through 48 hours) mice with burninjury alone demonstrated a depressed left ventricular fractionalshortening percentage (FS %) of 38.6+/−1.8% (Sham FS % 56.0+/−2.6%).Anti-MIF treated mice demonstrated a delayed improved cardiac functionafter burn injury, with complete recovery of function by 24 hours. Thisdemonstrates that the cytokine MIF mediates late burn injury associatedcardiac dysfunction, and also demonstrates that MIF is a pharmacologictarget for the treatment of burn injury associated cardiac dysfunctionas well as other MIF mediated complications such as ARDS associated withburn injury.

After burn injury, MIF is maximally released into the serum by 4 hours(FIG. 9); tissue release occurs to maximum by 8 hours (heart and spleen)(FIGS. 7 & 8). When MIF is neutralized by antibodies, cardiac functionimproves significantly by 12 hours and completely by 24 hours,demonstrating MIF modulation as a mediator of cardiac dysfunction invivo following burn injury (FIG. 12).

In a mouse model of burn injury, it has been demonstrated that LPSmediates the associated cardiac dysfunction through its interaction withthe Tlr-4 (toll-like receptor 4) and its interaction with IRAK-1. Thesource of LPS is believed to be gut-derived due to several potentialinsults associated with burn injury. These include intestinal ischemia,bacterial translocation, and increased intestinal permeability. Whilenot wishing to be bound by theory, it is thought that the production andrelease of inflammatory factors becomes systemic through gut associatedlymphoid tissues.

In a model of sub-lethal endotoxicosis in mice (4 mg/kg LPS), it isobserved that MIF mediated late cardiac depressant effects in vivo. Itis also determined that recombinant MIF induces an immediate cardiacdepression ex vivo by Langendorff assay in an LPS-independent manner.With respect to burn injury, one embodiment of the present inventionsuitably has several advantages compared to observations made in a modelof endotoxicosis. First, MIF is released at an earlier time point in theburn model (4 hours, FIG. 7) compared to the LPS challenge (8 hours) andthis increase in systemic MIF concentrations is significantly higher(2.2 fold increase (FIG. 9) vs. 1.5 fold). Secondly, the degree ofcardiac dysfunction was not as great in the burn injury model comparedto the endotoxicosis model as measured by echocardiography. With respectto burn injury, the present inventors desirably allows fractionalshortening percent (an estimate of cardiac output) to be decreased 38%from baseline by four hours (56.2 FS %−34.8 FS %/56.2 FS %) compared to53.7% in the endotoxicosis model (67.2 FS %−31.1 FS %/67.2 FS %) at fourhours. MIF inhibition in accordance with one embodiment of the presentinvention results in complete cardiac protection by 24 hours (FIG. 12),which did not occur until 48 hours in the endotoxicosis model, althoughsignificant protection was first seen by 8 hours in the endotoxicosismodel, and not until 12 hours in the burn injury model. Thisdemonstrates that MIF plays a greater role in burn injury associatedcardiac dysfunction. Lastly, the increase in cardiac MIF mRNA levels wasdetermined to be significantly increased by 8 hours in burn injury (FIG.10) and was not increased until 48 hours in the endotoxicosis model.While the cardiac effects of burn injury have been shown to be relatedto gut derived LPS, the effective insult is more complex than theendotoxicosis model in that it involves the skin as well as the gut andis a more physiologically relevant disease processes.

Neutralization of MIF resulted in cardioprotection starting at 12 hours,and marked cardiac dysfunction was identified in mice with burn injuryas early as four hours (FIG. 12). TNF-α, IL-1β, IL-6, and IL-10 aresecreted by cardiomyocytes and TNF-α and IL-1β are the primary mediatorsof the myocardial depression. The present invention contemplates therole these play in early cardiac dysfunction occurring before MIFmediated cardiac dysfunction. Since MIF is released locally from theheart just prior (8 hours) to the protective effects of anti-MIFtreatment (12 hours) seen by echocardiography, the present inventioncontemplates that MIF plays a significant role in the cardiacdysfunction seen at later time points in this model (12-48 hours afterburn injury). Of the ten additional cytokines investigated systemically(FIG. 9), only systemic IL-6 and IL-12 were modulated after burn injury.Systemic IL-12 levels were significantly decreased the first 12 hours,as has been reported in post-surgical sepsis patients. Decreases in thisTH1 cytokine is hypothesized as one mechanisms by which survival insepsis is decreased by impaired innate immune responses to infectionwhich has been identified in burn injury.

Major burn injury has been shown to produce significant increases inplasma and cardiac malondialdehyde (MDA) levels, a major product oflipid peroxidation that results from oxidative stress in tissues.Moreover, antioxidant therapy has been shown to decrease the release ofinflammatory cytokines in burn injury linking inflammatory responses toincreased oxidative stress. Previous studies have demonstrated that MIFis secreted from cardiomyocytes after oxidative stress, and, withoutwishing to be bound by theory, this may be one mechanism by which MIF isreleased. Oxidative stress initiated by H₂O₂ results in the activationof ERK1/2 signaling pathway and protein kinase C, the latter of whichappears to be responsible for the secretion of MIF. Therefore, thecardiac release of pre-formed MIF may be initiated with increases inoxidative stress in the heart which signals the release of MIF (FIGS. 7and 8) and upregulates its transcription (FIG. 10) in order to replenishthe stores in cardiomyocytes.

Most cytokines have tightly controlled expression that is upregulatedafter stimulation. MIF, however, exists preformed in substantial amountsand its expression relies not only on de novo protein synthesis, butalso from pre-existing stores which are controlled by secretorymechanisms involving ABC transporters. The MIF gene does not encode foran N-terminal signal sequence whose role is to translocate it to theendoplasmic reticulum. MIF is located predominantly in the cytosol insmall vesicles and the nucleus which are pinched off and released to theoutside of cells. Necrotic cell damage therefore leads to a release ofthe pre-stored MIF. While not wishing to be bound by theory, sinceprevious studies have clearly demonstrated that burn injury involvesskin necrosis in our model, MIF release may be directly released fromnecrotic cells of the skin, since MIF has been identified in the skinand localized to the basal layers of the epidermis.

In addition to the necrotic release of pre-formed MIF, damaged epidermisand fibroblasts have been shown to increase the expression and secretionof MIF. For example, in atopic dermatis, MIF is upregulated and plays apivotal role in the pathophysiology of the disease. Total body UV Bexposure in vivo has been shown to increase MIF production, suggestingits involvement in tissue injuries. Injured epidermis and culturedfibroblasts also increase the expression of MIF which contributespositively to the wound healing process. Systemic levels of MIF mayincrease more quickly and dramatically (2.2 fold by 4 hours in the burninjury model vs. 1.5 fold increase by 8 hours in the endotoxicosismodel) in this burn injury model compared to the endotoxicosis model dueto factors involving MIF released from burn injured skin.

MIF has been shown has been hypothesized to play a role in ARDS and lungcomplications of sepsis. Anti-MIF therapy has been shown to decreasepulmonary neutrophil accumulation in acute lung injury associated withsepsis. MIF is expressed in alveolar capillary endothelium andinfiltrating macrophages from ARDS patients. MIF expression has beenshown to form an amplifying loop with TNF-α effectively linking severeinflammation to these two cytokines in ARDS. Since ARDS is an importantand common complication of burn injury, the present inventioncontemplates anti-MIF therapies that are useful in other than cardiacprotective indications, and seriously affect outcomes.

MIF is unique among cytokines because it has multiple enzymaticactivities including oxidoreductase and tautomerase activity. Inhibitionof its tautomerase activity has been shown to counteract known MIFactivities such as its glucocorticoid override activities.Pharmacological inhibitors of MIF tautomerase activity have beendeveloped for diseases anti-MIF therapies have been effective such assepsis, asthma, atopic dermatitis, and acute respiratory syndrome(ARDS).

The cytokine MIF plays a significant role in the late cardiacdysfunction associated with burn injury. MIF itself is a direct cardiacdepressant and has a delayed release from the heart. The delayed releaseof MIF and development of inhibitors that potentially inhibit theactivity of MIF make MIF a potential target for diseases such as burninjury associated with morbidity and mortality related to itscardio-pulmonary effects.

In another embodiment of the invention, the present inventors have foundthat MIF release from the heart, liver, and spleen is dependent upon TNFreceptor I/II signaling after LPS challenge. Additionally, the presentinventors identify TNF receptor I/II signaling independent release intothe serum of MIF. Without TNF receptor signaling, MIF levels appearslightly delayed (12-24 hours compared to 8 hours in wild type) andslightly increased (1.7-2.3 fold baseline compared to 1.5 increase inwild type mice). Moreover, the TNF receptor independent MIF release inTNF receptor I/II deficient mice (TNFR−/−) is sufficient to mediatecardiac dysfunction by at least 24 hours after LPS challenge despite thelack of MIF release from tissues which has been previously identified inwild type mice. Early cardiac dysfunction was identified in the TNFR−/−mice and, without wishing to be bound by theory, is believed to belikely due to known mediators that the present inventors have identifiedto be highly expressed in this model (IL-1β, IL-6) in addition to otherpotential mediators (e.g. IL-18 as well as other mediators). In isolatedhearts (Langendorf prep), MIF was determined to induce an immediate(within 20 minutes) cardiac dysfunction (systolic and diastolic)directly in both TNFR−/− and wild type mice to the same extent. LPSinduced cardiac dysfunction in the TNFR−/− mice, however, was completelyablated by 48 hours with MIF neutralization with antibodies indicatingthat TNF receptor mediated independent release of MIF was capable ofinducing a profound late cardiac dysfunction (24 and 48 hours) in amodel of endotoxicosis.

The cytokine MIF is constitutively expressed in numerous cell typesincluding lymphocytes, macrophages, and the anterior pituitary. Manytissues also contain MIF including the heart, lung, liver, adrenal,spleen, kidney, skin, muscle, thymus, skin, and testes. The mechanism ofsecretion has recently been described in LPS stimulated monocytes.Inhibitors of classical protein secretion such as monensin or brefeldingA do not inhibit the secretion of MIF, suggesting a non-classicalprotein export route. When inhibitors of ABCA1 (ATP binding cassette A1)transporters (glyburide and probenicide) were given, MIF secretion didnot occur. MIF is located predominantly in the cytosol in small vesiclesand the nucleus which are pinched off and released to the outside ofcells. This non-classical, vesicle-mediated secretory pathway has beenshown to be a mechanism of secretion of other important inflammatorymediators such as HMGB1, which has been shown to play a significant rolein inflammatory diseases and specifically sepsis. In the presentinvention, the dependence of MIF secretion on TNF-α signaling in severaltissues is described for the first time.

MIF has numerous biological activities including glucocorticoidantagonist properties, catalytic properties which are regulated throughthe coactivator JAB1/CSN5 and the cell surface protein CD74/Ii chain.Specific secretion of MIF results after inflammatory stimuli such asendotoxin (LPS) and tumor necrosis factor, as well as hormones such asACTH, and angiotensin II. In addition to immune cells, endocrine cellsand some epithelial cells secrete MIF. Secretion is due to anenhancement of MIF expression and de novo synthesis as well as aninduction of the release from pre-existing stores; both of which havebeen previously demonstrated in the heart.

Cardiac MIF has been reported to be released maximally at 12 hours afterLPS challenge in wild type mice. Serum MIF levels in wild type miceafter LPS challenge maximally release at 8 hours, corresponding to earlyprotection of cardiac dysfunction. When TNF-α signaling is inhibitedafter LPS challenge by either Enbrel®(D pretreatment or in TNFR−/− mice,the MIF levels peak later and slightly higher, indicating that TNF-α hassome control over serum MIF levels, but does not inhibit MIF release.When wild type mice were pretreated with anti-MIF neutralizingantibodies and subjected to an LPS challenge (4 mg/kg), an initialsevere cardiac dysfunction was seen at four hours that was identical tomice given LPS alone. However, initial significant protection was seenat 8 hours and improved until 48 hours where cardiac function was notsignificantly different from control animals. Since MIF had a nearlyimmediate and direct cardiac effect in isolated wild type hearts, it wasbelieved that MIF played a role in cardiac function which paralleled itsdelayed release. However, in the present invention it is demonstratedthat non-cardiac release of MIF can have significant effects on functionin vivo as well (24 and 48 hours). These effects occur later thanpreviously described (initial protection at 8 hours) and parallel adelay in MIF release systematically when TNF-α signaling is blocked(12-24 hours) compared to wild type mice (maximum MIF release at 8hours).

It is believed that the early cardiac dysfunction can be attributed toseveral cytokines which have been shown to mediate sepsis (and LPS)associated cardiac dysfunction such as TNF-α, IL-1β, and IL-18. Atemporal discordance between myocardial TNF-α levels and contractiledysfunction in endotoxemia has been reported. These findings of LPSassociated myocardial dysfunction did not occur until TNF-α levelsreturned to baseline contradict other findings which have reportedprotection from LPS induced cardiac dysfunction after the neutralizationof TNF-α. These findings and others have led to the hypothesis thatTNF-α is required for LPS induced increases of downstream mediators suchas IL-1β, IL-6, IL-18 among other not yet identified factors. Thepresent inventors demonstrate that TNF signaling mediates some (MIFsecretion from heart, liver, spleen), but not all, of the effects of MIFin their model of endotoxemia.

The sentinel role of TNF-α signaling has been studied by otherinvestigators, specifically in relationship to the cytokine IL-18 inendotoxemia models. After LPS challenge in TNF-α knockout (−/−) mice,IL-18 levels in the heart are not significantly changed, while wild typemice demonstrated significant increases in IL-18 levels. When IL-18 isneutralized, this study demonstrated that the LPS induced cardiacdysfunction is reduced and that IL-18 appears to have downstream effectson tissue TNF-α, IL-1β, as well as ICAM-1/VCAM-1 levels. While thisstudy focused on the myocardial production and release of IL-18 in themyocardium, non-cardiac sources of IL-18 were not investigated. Thisstudy is believed to be similar to that of the present inventors in theTNR-α dependence on tissue (cardiac) production/release of IL-18.

Similarly, it is important to note a previous study that identified theeffects of TNF-α inhibition on myocardial cytokine mRNA expression aswell as plasma cytokine levels after LPS challenge. Inhibition of TNFpartially but significantly reduced plasma levels of IL-1, IL-6, andMCP-1 but not MIF, TNF, IL-10, or IL-12 at 2 hours; in contrast anti-TNFpretreatment significantly reduced myocardial expression of IL-1β butnot other cytokines including MIF at this early time point. Since thepresent inventors have shown that MIF is shown to release later than 2hours (peaks at 12 hours), the present inventors describe the TNFindependent and TNF dependent MIF pathways for the first time thatparallel IL-1β, IL-6, and MCP-1 in this previous study. These areimportant findings since most of these cytokines have been shown todirectly mediate cardiac dysfunction.

While the cardiac dysfunction of the TNFR−/− mice after LPS challenge inthis study (18.2% (45.9-27.7 FS %) is not as profound as that determinedin wild type mice in our previous study 36.7% (67.3-30.6), thedepression is significant. Since multiple cytokines have been attributedto the early cardiac dysfunction after LPS challenge, we determinedserum cytokine levels in the TNFR−/− model. The levels of TNF-α wasdramatically increased compared to wild type mice, however, with nofunctional receptors (TNF receptor I or TNF receptor II), it effectswere not mediating the early cardiac dysfunction seen. However,increases in IL-1β and IL-6 were clearly seen at 4 hours (31.2 and 8.5fold above wild type mice, respectively) and likely contributed to earlycardiac dysfunction in addition to other cytokines such as IL-1βrecently described as well as other mediators not yet described. Therole of TNF-α signaling in the regulation of TNF-α secretion haspreviously been described. However, the present inventors havedemonstrated for the first time that TNF-α receptor signaling regulatesIL-1β, IL-12, and IL-10 by a negative feedback mechanism and positivelyregulates IFN-γ (FIG. 19). Similarly, mice deficient in IL-6 haveaugmented expression of IL-1β and TNF-α after LPS challenge and thepresent inventors contemplate that cardiac IL-6 suppresses theexpression of proinflammatory mediators including itself by a negativefeedback mechanism.

TNF-α signaling occurs through 2 receptors, TNF-α receptor 1 and 2.These two pathways have divergent signaling pathways. The interaction ofTNF-α and receptor 1 activates several signal transduction pathwaysincluding NF-KB, which the TNF-α receptor does not. To investigate thewhich of the two possible TNF receptors were responsible for theinhibition of MIF release in the heart, the present inventors challengedmice in which IkB overexpression in the heart resulted in nearlycomplete NF-kB inhibition. By western analysis, the present inventorsdemonstrated that no release occurred in the same manner as the TNFR−/−mice at all time points tested (data not shown, identical to FIG. 2A).These mice have circulating TNF-α equivalent to wild types (since NF-kBinhibition is cardiac specific). Additionally, these mice express MIF inthe serum similar to wild type mice. Accordingly, the present inventioncontemplates that the TNF receptor 1 may mediate the tissue release seenin wild type mice from the heart. Since the phenotype of this heart iscompletely protected after LPS challenge by echo during the first 48hours (data not shown), the present inventors contemplate thatcirculating MIF requires upstream NF-kB mediated proteins to be signal(TNF-α, IL-1β) or that MIF mediates its effects by NF-kB itself.

The release of IL-10 and IFN-γ after LPS challenge in wild type mice isattenuated when they are pre-treated with MIF neutralizing antibodies(FIG. 20). Since MIF has a delayed release, these are likely thecytokines affected due to their temporal relationship to MIF, occurringafter MIF is released (FIG. 13). These findings indicate that MIFactivity plays a significant role in IL-10 and IFN-γ release afterendotoxin challenge, although it is likely not the only signal for theirrelease. It is interesting to note that in the TNFR−/− mice (FIG. 19C)that IFN-γ release is inhibited as well, making both TNF and MIFnecessary for its release. Similarly, in a model of dextransulfate-induced colitis by anti-MIF antibodies, IFN-γ was signficantllysuppressed. The expression of iNOS in cardiac myocytes have been shownto expressed when TNF-α and LPS are given with IFN-γ, but NOT withoutIFN-γ. Since iNOS itself plays a role in the regulatory pathways of LPSassociated cardiac dysfunction, these cytokine pathways are complex andlikely interact closely.

Efforts at inhibiting TNF-α in order to reduce morbidity and mortalityin sepsis and septic shock have previously failed clinically, eventhough pre-clinical studies in mice protected against endotoxinchallenges. The present inventors have found that the inhibition ofTNF-α signaling prior to LPS challenge results in the inhibition of MIFsecretion from the tissues, although serum MIF release is unaffected.Moreover, when Enbrel was given just immediately prior to or after LPSchallenge instead of 90 minutes prior, tissue release of MIF occurredsporatically (data not shown) which may account for additionallydownstream MIF effects by this therapy. While not wishing to be bound bytheory, these findings indicate mechanisms by which anti-TNF-α therapiesmay not have worked due to MIF effects.

While this study of LPS challenge in the absence of a live infectioncannot be directly extrapolated to sepsis-induced myocardialdysfunction, it is important to note that MIF has been shown to bephysiologically relevant in a model of live polymicrobial peritonitis(CLP). When MIF neutralizing antibodies were given to TNF-α−/− mice(particularly susceptible to CLP insult) MIF was protective againstlethality (62% survival at 9 days in MIF neutralized mice compared to 0%with CLP only) at 15 hours. Similarly, in wild type mice, MIFneutralizing antibodies were protective for 9 days (endpoint ofexperiment) where 81% survived compared to 31%, even when antibody wasgiven up to 4.5 hours after the insult (61% survival compared to 5%).Accordingly, the present inventors contemplate anti-MIF therapiesagainst sepsis-induced myocardial dysfunction.

Since MIF release is delayed and partially unaffected by blocking ofimportant upstream mediators such as TNF-α, it may represent one goodtherapeutic target. To this end, the inhibition of MIF's tautomeraseactivity that mediate some of its biological functions may be intervenedpharmacologically. Moreover, recent non-classical secretory mechanismsof release also are potential target for therapy. However, it isimportant to realize that other significant targets in sepsis that areknown (HMGB1) as well as undescribed targets still exist. Therefore, itis important to understand the effects of therapeutic invention of allof these mediators.

The present inventors have demonstrated that MIF plays a significantrole in LPS induced cardiac dysfunction which is believed to contributeto myocardial dysfunction during sepsis. Because TNF-α is thought to bean important sentinel cytokine in LPS induced cardiac dysfunction, theeffects of blocking TNF-α signaling pathways in vivo on MIF inducedcardiac dysfunction have been investigated. Serum concentrations and thetemporal distribution of MIF was slightly increased and delayed by theinhibition of TNF-α signaling (maximally increased 12-24 hours (1.7-2.3fold baseline with TNF-α signaling inhibition vs. 8 hours in wild type(1.5 fold increase). The release of pre-formed MIF from heart, liver,and spleen did not occur after LPS challenge after inhibition of TNF-αsignaling unlike the delayed maximum release at 12 hours seen previouslyin wild type mice. Northern analysis of cardiac MIF mRNA revealed nosignificant changes in transcription after LPS challenge. Whenrecombinant MIF was applied to isolated TNFR−/− hearts (Langendorffpreparation), a significant decrease in cardiac function was detectedequal to wild type mice, indicating that MIF signaling was TNF-α.Echocardiography of TNFR−/− mice after LPS challenge demonstrated anearly cardiac dysfunction (18.2% decrease in fractional shortening %)that minimally improved after 48 hours. When MIF was neutralized bymonoclonal antibodies, cardiac function significantly improved by 24hours and completely recovered by 48 hours, indicating that MIF played arole in late LPS associated cardiac dysfunction despite the lack oftissue release. This study demonstrates for the first time that MIFrelease from cardiac and other tissues is dependent upon TNF signaling,and serum release of MIF is unaffected by a lack TNF signaling after LPSchallenge and is adequate to mediate cardiac dysfunction.

Macrophage migration inhibitory factor (MIF) is pluripotent cytokinewith direct and significantly deleterious effects on heart functionduring sepsis (severe infections). One embodiment of the presentinvention demonstrates in a mouse model that the inhibition of MIFactivity can profoundly improve cardiac function following acutemyocardial infarction (See FIGS. 21-25). This improvement is evidentwithin hours, and lasts for the duration of the experiment (1 week). Itis highly likely that modulation of MIF will decrease infarct size andother pathologic parameters. The degree of improvement in cardiacfunction is remarkable, and substantially in excess by at least 10 foldcompared to other immune targets such as TNF-alpha.

Prior to the present invention there were no treatments for myocardialinfarction which target chemical/cytokine mediators of heart dysfunctionand acute myocardial tissue injury. Current technology aims to minimizeinfarction by therapies such as TPA. Once TPA or similar therapy isgiven, there are no drug therapies to prevent cardiogenic shock/failureaside from standard inotropes (dobutamine) or mechanical devices(intra-aortic balloon pump). Therapies to block MIF being givensimultaneously with TPA or balloon angioplasty to further improve boththe short term as well as the long term heart function, and potentiallyminimize infarct size are contemplated herein.

One embodiment of the present invention solves the problems of acute andchronic heart dysfunction following acute myocardial infarction. Thepresent invention makes it possible to provide a unique class oftherapies in that it modulates an immune mediator, i.e., MIF. Withoutwishing to be bound by theory, it is believed that the present inventionmay directly decreases infarct size. There is no equivalent therapy topreserve long term cardiac function. For short term function, modulationof MIF activity in accordance with the present invention would minimizethe need for intra-aortic balloon pumps and other mechanical devices.Suitable monoclonal anti-MIF antibodies are obtained from CytokinePharmaScience, Inc., King of Prussia, Pa.

Another embodiment of the present invention relates to inhibition ofCD74 to protect cardiodysfunction associated with severe disease such assepsis, trauma, acute MI, and congestive heart failure. While CD74 hasbeen described on circulating immune cells and antigen presenting cells(in association with MHC Class II), until the present invention, thepresence of CD74 in the heart (as well as other organs) has not beenreported. More importantly, the function role of CD74 on cardiacfunction in physiological or disease processes has not previously beendemonstrated. While CD74 has been shown to mediate MIF activity invitro, this has not been confirmed independently and is restricted tofibroblasts and leukocytes.

Suitable anti-human CD74 antibodies are available, for example, from BDBiosciences (product catalog numbers 555538 (Clone M-B741; FormatPurified; Isotype Mouse IgG_(2a), κ; W.S. No V CD74.4; Reactivity Human)and 555612 (Clone LN2; Format Purified; Isotype Mouse IgG₁, K; W.S. No VCD74.3; Reactivity Human).

The present invention makes it possible to use anti-cytokine therapy(including anti-MIF) in cardiac diseases. Preliminary experiments usinganti-TNF therapy in sepsis models did not work. Additionally, MIF, theputative cytokine blocked by CD74 inhibition, is a cytokine that occurslater, and in our model of acute MI is increased for several weeks afterthe insult, allowing for intervention during any of that time.

Cardiac dysfunction early in acute MI and sepsis accounts for the highmorbidity and mortaility associated with each of these diseases. Thepresent inventors contemplate cytokine therapy that can enhanceperformance and potentially lower the morbidity associated with each.

Most therapeutic interventions in cardiac disease have focused onreperfusion (MI) or inotropes (MI and sepsis). By one embodiment of thepresent invention, intervening with the CD74 receptor, the putativereceptor for MIF, both acute and chronic cardiogenic impairment may beattenuated and improve survival/outcomes.

MIF is secreted from cardiomyocytes following LPS challenge, anddirectly mediates a late onset (>6 hours) cardiac dysfunction. In immunecells, CD74 was determined to be the MIF receptor, exerting effects viaERK1/2 intracellular signaling pathways. To determine if CD74 mediatesMIF-induced cardiac dysfunction in sepsis, the present inventorschallenged: 1) wild type mice (C57BL/6) with LPS; 2) wild type micepre-treated with anti-CD74 monocolonal neutralizing antibodies; andchallenged with LPS, and 3) CD74 knock-out mice with LPS (4 mg/kg).Serial echocardiography was performed and fractional shortening (FS %)was determined. At 24 hours, significant dysfunction was observed in WTmice given LPS (FS %=31.6%±3.3%) compare to controls (FS %=58+1%). Inboth anti-CD74 antibody treated and CD74 knock-out mice challenged withLPS, cardiac function was significantly improved compared to wild typemice given LPS alone (FS %=49+3.6% and 53.3±2.4%, respectively, p<0.05).As CD74 expression has never been documented in the heart, the presentinventors performed immunoblots and histochemistry which confirmed thatCD74 was constituitively present on cardiac cell membranes and in thecytosol; and was substantially regulated after LPS challenge (nearlyabsent at 12 hours->4 fold decrease). The present inventors demonstratethat CD74 is expressed on cardiomyocytes and is a critical mediator ofcardiac dysfunction.

Another embodiment of the present invention relates to the inhibition ofMIF activity by use of one or more soluble MIF receptor or MIF receptorantagonist. As an example, with anti-TNFα therapies, REMICADE™ orINFLIXIMAB™ (antibody TNFα) and ENBREL™ or ETANERCEPT™ (solubleTNF-receptor) are suitable. This method includes administering one ormore of the soluble MIF receptor and/or MIF receptor antagonist in aneffective amount for treating and/or preventing cardiac dysfunction in asubject in need thereof, treating and/or preventing irregularity inmyocardial activity in a subject in need thereof, treating and/orpreventing depression in myocardial activity in a subject in needthereof, treating and/or preventing burn-injury associated cardiacdysfunction in a subject in need thereof, treating and/or preventingcardiac dysfunction following acute myocardial infarction in a subjectin need thereof, treating and/or preventing cardiodepression in asubject in need thereof, or a combination thereof to a subject in needthereof.

Another embodiment of the present invention relates to the use of smallmolecule MIF inhibitors (sometimes called “MIF antagonists” or“isoxazoline compounds”) in treating and/or preventing cardiacdysfunction in a subject in need thereof, treating and/or preventingirregularity in myocardial activity in a subject in need thereof,treating and/or preventing depression in myocardial activity in asubject in need thereof, treating and/or preventing burn-injuryassociated cardiac dysfunction in a subject in need thereof, treatingand/or preventing cardiac dysfunction following acute myocardialinfarction in a subject in need thereof, treating and/or preventingcardiodepression in a subject in need thereof, or a combination thereof.

In the following chemical formulae, the use of the superscript on asubstituent is to identify a substituent name (e.g., “R²” is used toindicate an R²-named substituent), while the use of a subscript is usedto enumerate the number of times a substituent occurs at that molecularsite (e.g., “R₂ “or “(R)₂” both are used to indicate two substituentssimply named as “R”).

A suitable small molecule MIF inhibitor for use in the methods hereinhas the following Formula I:

wherein:

-   -   R₁₋₄ are, independently, R, halo, N₃, CN, OH, NRR′, or SH;    -   R and R′ are, independently, H or C₁₋₆ alkyl;    -   X is R, halo, N₃, CN, OR, NRR′, SH, ═O, ═CH₂, or A;    -   A is a substituted or unsubstituted aromatic ring;    -   Y is R, NRR′, NRR″ or (CH₂)_(n)-A;    -   Z is R, OR, OR″, NRR′, NRR″, or A;    -   R″ is a saturated or unsaturated, straight or branched chain        C₂-C₁₈;    -   and n is 0 or 1.

Preferably, the compound of Formula I is ap-hydroxyphenyl-isoxazoline-containing compound, wherein each of R,R₁₋₄, X and Y is H or —CH₂-A, and Z is OR. More preferably, the compoundof Formula I is an ester of(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic, particularlythe acid methyl ester thereof (sometimes identified as “ISO-1” or “CPSI”or “CPSI-26” herein) which is also known as p-hydroxyphenol-isoxazolinemethyl ester. Still more preferably the compound is an ester of2-{3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl}-3-phenyl-propinoicacid, particularly the methyl ester thereof (identified as “ISO-2”).

Other suitable small molecule MIF inhibitors for use in the methodherein have the following Formulas II or III:

-   -   wherein B is either oxygen or sulphur and each “R” is        independently defined:    -   with the requirement that each “R” cannot only occur as hydrogen        on either Formula II or III (i.e., at least one R on either        Formula II or III is an “R” substituent other than hydrogen),        and any B is independently either oxygen or sulphur; any R¹ is        independently hydrogen, (C₁-C₆)alkyl or some other suitable        substituent, any R² is an amine, an alkoxy or some other        suitable substituent; and “m” is independently either zero or an        integer from one to twenty;    -   each X is independently either carbon or nitrogen; and when any        X is carbon, then Y is the substituent defined independently for        each X as    -   each Z is independently either hydrogen, hydroxyl, halogen, or        some other suitable substituent; and    -   “in” is independently any of 0, 1, 2, 3, or 4;    -   and pharmaceutically acceptable salts and prodrugs thereof.

The present invention also relates to the pharmaceutically acceptableacid addition salts of the compounds of general Formulas I, II, or III.The acids which are used to prepare the pharmaceutically acceptable acidaddition salts of the aforementioned base compounds of this inventionare those which form non-toxic acid addition salts, i.e., saltscontaining pharmacologically acceptable anions, such as the chloride,bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,acetate, lactate, citrate, acid citrate, tartrate, bitartrate,succinate, maleate, fumarate, glutamate, L-lactate, L-tartrate,tosylate, mesylate, gluconate, saccharate, benzoate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate))salts.

The invention also relates to base addition salts of the small moleculeMIF inhibitors. The chemical bases that may be used as reagents toprepare pharmaceutically acceptable base salts of those compounds ofgeneral Formulas I, II, or III that are acidic in nature are those thatform non-toxic base salts with such compounds. Such non-toxic base saltsinclude, but are not limited to those derived from suchpharmacologically acceptable cations such as alkali metal cations (e.g.,potassium and sodium) and alkaline earth metal cations (e.g., calciumand magnesium), ammonium or water-soluble amine addition salts such asN-methylglucamine-(meglumine), and the lower alkanolammonium and otherbase salts of pharmaceutically acceptable organic amines.

The compounds and prodrugs of the small molecule MIF inhibitors canexist in several tautomeric forms, and geometric isomers and mixturesthereof. All such tautomeric forms are included within the scope of thepresent invention. Tautomers exist as mixtures of tautomers in solution.In solid form, usually one tautomer predominates. Even though onetautomer may be described, the present invention includes all tautomersof the small molecule MIF inhibitors.

The present invention also includes atropisomers of the small moleculeMIF inhibitors. Atropisomers refer to compounds of the small moleculeMIF inhibitors that can be separated into rotationally restrictedisomers. The small molecule MIF inhibitors may contain olefin-likedouble bonds. When such bonds are present, the small molecule MIFinhibitors exist as cis and trans configurations and as mixturesthereof.

A “suitable substituent” is intended to mean a chemically andpharmaceutically acceptable functional group i.e., a moiety that doesnot negate the inhibitory activity of the small molecule MIF inhibitors.Such suitable substituents may be routinely selected by those skilled inthe art. Illustrative examples of suitable substituents include, but arenot limited to halo groups, perfluoroalkyl groups, perfluoroalkoxygroups, alkyl groups, alkenyl groups, alkynyl groups, hydroxy groups,oxo groups, mercapto groups, alkylthio groups, alkoxy groups, aryl orheteroaryl groups, aryloxy or heteroaryloxy groups, aralkyl orheteroaralkyl groups, aralkoxy or heteroaralkoxy groups, HO—(C═O)—groups, amino groups, alkyl- and dialkylamino groups, carbamoyl groups,alkylcarbonyl groups, alkoxycarbonyl groups, alkylaminocarbonyl groupsdialkylamino carbonyl groups, arylcarbonyl groups, aryloxycarbonylgroups, alkylsulfonyl groups, arylsulfonyl groups and the like.

Preferred small molecule MIF inhibitors may be found in U.S. provisionalapplication 60/556,440, filed Mar. 26, 2004, U.S. provisionalapplication 60/296,478, filed Jun. 8, 2001; and U.S. application Ser.No. 10/164,630, filed Jun. 10, 2002, the entire contents of each ofwhich is hereby incorporated by reference for all purposes.

Preferably, in this embodiment, an effective amount of one or more smallmolecule MIF inhibitors and/or salts thereof is administered as activeingredient to a subject in need thereof. Combinations of small moleculeMIF inhibitors are also possible.

The small molecule MIF inhibitor compounds can also be administered inform of their pharmaceutically active salts and/or prodrugs asappropriate. Combinations of salts and/or prodrugs are possible, as arecombinations of salt-forms and non-salt-forms of the small molecule MIFinhibitor.

Another embodiment of the present invention relates to pharmaceuticalcompositions suitable in treating and/or preventing cardiac dysfunctionin a subject in need thereof, treating and/or preventing irregularity inmyocardial activity in a subject in need thereof, treating and/orpreventing depression in myocardial activity in a subject in needthereof, treating and/or preventing burn-injury associated cardiacdysfunction in a subject in need thereof, treating and/or preventingcardiac dysfunction following acute myocardial infarction in a subjectin need thereof, treating and/or preventing cardiodepression in asubject in need thereof, or a combination thereof, which includes one ormore small molecule MIF inhibitors and/or salts thereof as activeingredient and at least one pharmaceutically acceptable carrier,excipient, adjuvent and/or diluent.

Another embodiment of the present invention relates to theadministration, to a subject in need thereof, of an effective amount ofa composition which includes at least one small molecule MIF inhibitor,salt thereof, and/or prodrug thereof and at least one anti-MIF antibodyin treating and/or preventing cardiac dysfunction in a subject in needthereof, treating and/or preventing irregularity in myocardial activityin a subject in need thereof, treating and/or preventing depression inmyocardial activity in a subject in need thereof, treating and/orpreventing burn-injury associated cardiac dysfunction in a subject inneed thereof, treating and/or preventing cardiac dysfunction followingacute myocardial infarction in a subject in need thereof, treatingand/or preventing cardiodepression in a subject in need thereof, or acombination thereof.

Another embodiment of the present invention relates to pharmaceuticalcompositions suitable in treating and/or preventing cardiac dysfunctionin a subject in need thereof, treating and/or preventing irregularity inmyocardial activity in a subject in need thereof, treating and/orpreventing depression in myocardial activity in a subject in needthereof, treating and/or preventing burn-injury associated cardiacdysfunction in a subject in need thereof, treating and/or preventingcardiac dysfunction following acute myocardial infarction in a subjectin need thereof, treating and/or preventing cardiodepression in asubject in need thereof, or a combination thereof, which includes aneffective amount of a combination of at least one small molecule MIFinhibitor, salt thereof, and/or prodrug thereof and at least oneanti-MIF antibody, and at least one pharmaceutically acceptable carrier,excipient, adjuvent and/or diluent.

Another embodiment of the present invention relates to theadministration, to a subject in need thereof, of an effective amount ofa composition which includes a combination of at least one smallmolecule MIF inhibitor, salt thereof, and/or prodrug thereof, at leastone anti-TNFR antibody and at least one anti-MIF antibody in treatingand/or preventing cardiac dysfunction in a subject in need thereof,treating and/or preventing irregularity in myocardial activity in asubject in need thereof, treating and/or preventing depression inmyocardial activity in a subject in need thereof, treating and/orpreventing burn-injury associated cardiac dysfunction in a subject inneed thereof, treating and/or preventing cardiac dysfunction followingacute myocardial infarction in a subject in need thereof, treatingand/or preventing cardiodepression in a subject in need thereof, or acombination thereof.

Another embodiment of the present invention relates to pharmaceuticalcompositions suitable in treating and/or preventing cardiac dysfunctionin a subject in need thereof, treating and/or preventing irregularity inmyocardial activity in a subject in need thereof, treating and/orpreventing depression in myocardial activity in a subject in needthereof, treating and/or preventing burn-injury associated cardiacdysfunction in a subject in need thereof, treating and/or preventingcardiac dysfunction following acute myocardial infarction in a subjectin need thereof, treating and/or preventing cardiodepression in asubject in need thereof, or a combination thereof, which includes aneffective amount of a combination of at least one small molecule MIFinhibitor, salt thereof, and/or prodrug thereof, at least one anti-TNFRantibody and at least one anti-MIF antibody, and at least onepharmaceutically acceptable carrier, excipient, adjuvent and/or diluent.

Another embodiment of the present invention relates to theadministration, to a subject in need thereof, of an effective amount ofa composition which includes a combination of at least one smallmolecule MIF inhibitor, salt thereof, and/or prodrug thereof, at leastone anti-CD-74 antibody and at least one anti-MIF antibody in treatingand/or preventing cardiac dysfunction in a subject in need thereof,treating and/or preventing irregularity in myocardial activity in asubject in need thereof, treating and/or preventing depression inmyocardial activity in a subject in need thereof, treating and/orpreventing burn-injury associated cardiac dysfunction in a subject inneed thereof, treating and/or preventing cardiac dysfunction followingacute myocardial infarction in a subject in need thereof, treatingand/or preventing cardiodepression in a subject in need thereof, or acombination thereof.

Another embodiment of the present invention relates to pharmaceuticalcompositions suitable in treating and/or preventing cardiac dysfunctionin a subject in need thereof, treating and/or preventing irregularity inmyocardial activity in a subject in need thereof, treating and/orpreventing depression in myocardial activity in a subject in needthereof, treating and/or preventing burn-injury associated cardiacdysfunction in a subject in need thereof, treating and/or preventingcardiac dysfunction following acute myocardial infarction in a subjectin need thereof, treating and/or preventing cardiodepression in asubject in need thereof, or a combination thereof, which includes aneffective amount of a combination of at least one small molecule MIFinhibitor, salt thereof, and/or prodrug thereof, at least one anti-CD-74antibody and at least one anti-MIF antibody, and at least onepharmaceutically acceptable carrier, excipient, adjuvent and/or diluent.

Another embodiment of the present invention relates to theadministration, to a subject in need thereof, of an effective amount ofa composition which includes a combination of at least one smallmolecule MIF inhibitor, salt thereof, and/or prodrug thereof and atleast one anti-CD-74 antibody in treating and/or preventing cardiacdysfunction in a subject in need thereof, treating and/or preventingirregularity in myocardial activity in a subject in need thereof,treating and/or preventing depression in myocardial activity in asubject in need thereof, treating and/or preventing burn-injuryassociated cardiac dysfunction in a subject in need thereof treatingand/or preventing cardiac dysfunction following acute myocardialinfarction in a subject in need thereof, treating and/or preventingcardiodepression in a subject in need thereof, or a combination thereof.

Another embodiment of the present invention relates to pharmaceuticalcompositions suitable in treating and/or preventing cardiac dysfunctionin a subject in need thereof, treating and/or preventing irregularity inmyocardial activity in a subject in need thereof, treating and/orpreventing depression in myocardial activity in a subject in needthereof, treating and/or preventing burn-injury associated cardiacdysfunction in a subject in need thereof, treating and/or preventingcardiac dysfunction following acute myocardial infarction in a subjectin need thereof, treating and/or preventing cardiodepression in asubject in need thereof, or a combination thereof, which includes aneffective amount of a combination of at least one small molecule MIFinhibitor, salt thereof, and/or prodrug thereof, at least one anti-CD-74antibody, and at least one pharmaceutically acceptable carrier,excipient, adjuvent and/or diluent.

Without wishing to be bound by theory, it is believed thatneutralization of tautomerase activity (which the cytokine MIF has beenshown to have) with small molecule MIF inhibitors results in orcontributes to the inhibition of cardiodepression that occurs aftermyocardial infarction. Inhibition of tautomerase with small moleculeinhibitors results in similar cardioprotection as anti-MIF antibodies.Since MIF has been shown to have tautomerase activity and it has beensuggested in in vitro studies to mediate its activity, it is believedthat neutralization of MIF may occur after its tautomerase activity isinhibited.

The present inventors have found that inhibiting MIF and/or neutralizingMIF tautomerase activity provides an anti-cytokine/inflammation therapyagainst cardiac diseases. Inhibition of MIF improves cardiac functionafter myocardial infarction and makes it possible to help with the acutesequelae of myocardial infarctions, such as reducing cardiac dysfunctionearly in acute myocardial infarction and reducing the associated highmorbidity and mortality. One advantage the present invention has overcurrent technologies is that unlike therapeutic interventions in cardiacdisease that focus on inotropes, the present invention, by treatingand/or preventing the cause of the cardiac dysfunction, both acute andchronic cardiogenic impairment may be attenuated and improvesurvival/outcomes.

One embodiment of the present invention relates to pharmaceuticalcompositions comprising at least one compound of the present inventionas an active ingredient (and/or salt and/or prodrug thereof) and atleast one pharmaceutically acceptable carrier, excipient, adjuventand/or diluent.

The compounds can also be administered in form of their pharmaceuticallyactive salts optionally using substantially nontoxic pharmaceuticallyacceptable carrier, excipients, adjuvants or diluents. The compositionsof the present invention may be prepared in any conventional solid orliquid carrier or diluent and optionally any conventionalpharmaceutically-made adjuvant at suitable dosage level in a known way.The preferred preparations are in administrable form which is suitablefor oral application. These administrable forms, for example, includepills, tablets, film tablets, coated tablets, capsules, powders anddeposits.

Forms other than oral administrable forms are also possible. Thecompounds of the present invention and/or pharmaceutical preparationscontaining said compounds may be administered by any appropriate means,including but not limited to injection (intravenous, intraperitoneal,intramuscular, subcutaneous) by absorption through epithelial ormucocutaneous linings (oral mucosa, rectal and vaginal epitheliallinings, nasopharyngial mucosa, intestinal mucosa); orally, rectally,transdermally, topically, intradermally, intragastrally, intracutanly,intravaginally, intravasally, intranasally, intrabuccally, percutanly,sublingually, or any other means available within the pharmaceuticalarts.

The pharmaceutical compositions of the present invention, containing atleast one compound of the present invention or pharmaceuticallyacceptable salts thereof as an active ingredient, will typically beadministered in admixture with suitable carrier materials suitablyselected with respect to the intended form of administration, i.e. oraltablets, capsules (either solid-filled, semi-solid filled or liquidfilled), powders for constitution, oral gels, elixirs, dispersiblegranules, syrups, suspensions, and the like, and consistent withconventional pharmaceutical practices. For example, for oraladministration in the form of tablets or capsules, the active drugcomponent may be combined with any oral nontoxic pharmaceuticallyacceptable inert carrier, such as lactose, starch, sucrose, cellulose,magnesium stearate, dicalcium phosphate, calcium sulfate, talc,mannitol, ethyl alcohol (liquid forms) and the like. Moreover, whendesired or needed, suitable binders, lubricants, disintegrating agentsand coloring agents may also be incorporated in the mixture.

Pharmaceutical compositions may be comprised of from about 5 to about 95percent by weight of the active ingredient, which range includes allvalues and subranges therebetween, including 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, and 95% by weight.

Suitable binders include starch, gelatin, natural sugars, cornsweeteners, natural and synthetic gums such as acacia, sodium alginate,carboxymethyl-cellulose, polyethylene glycol and waxes. Among thelubricants there may be mentioned for use in these dosage forms, boricacid, sodium benzoate, sodium acetate, sodium chloride, and the like.Disintegrants include starch, methylcellulose, guar gum and the like.Sweetening and flavoring agents and preservatives may also be includedwhere appropriate. Some of the terms noted above, namely disintegrants,diluents, lubricants, binders and the like, are discussed in more detailbelow.

Additionally, the compounds or compositions of the present invention maybe formulated in sustained release form to provide the rate controlledrelease of any one or more of the components or active ingredients tooptimize the therapeutic effects, i.e. antihistaminic activity and thelike. Suitable dosage forms for sustained release include layeredtablets containing layers of varying disintegration rates or controlledrelease polymeric matrices impregnated with the active components andshaped in tablet form or capsules containing such impregnated orencapsulated porous polymeric matrices.

Liquid form preparations include solutions, suspensions and emulsions.As an example may be mentioned water, ethanolic, water-ethanol orwater-propylene glycol solutions for parenteral injections or additionof sweeteners and opacifiers for oral solutions, suspensions andemulsions. Liquid form preparations may also include solutions forintranasal administration.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be in combination with apharmaceutically acceptable carrier such as inert compressed gas, e.g.nitrogen.

For preparing suppositories, a low melting wax such as a mixture offatty acid glycerides such as cocoa butter is first melted, and theactive ingredient is dispersed homogeneously therein by stirring orsimilar mixing. The molten homogeneous mixture is then poured intoconvenient sized molds, allowed to cool and thereby solidify.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions and emulsions.

The compounds of the present invention may also be deliverabletransdermally. The transdermal compositions may take the form of creams,lotions, aerosols and/or emulsions and can be included in a transdermalpatch of the matrix or reservoir type as are conventional in the art forthis purpose.

The term capsule refers to a special container or enclosure made ofmethyl cellulose, polyvinyl alcohols, or denatured gelatins or starchfor holding or containing compositions comprising the activeingredients. Hard shell capsules are typically made of blends ofrelatively high gel strength bone and pork skin gelatins. The capsuleitself may contain small amounts of dyes, opaquing agents, plasticizersand preservatives.

Tablet means compressed or molded solid dosage form containing theactive ingredients with suitable diluents. The tablet can be prepared bycompression of mixtures or granulations obtained by wet granulation, drygranulation or by compaction well known to a person skilled in the art.

Oral gels refers to the active ingredients dispersed or solubilized in ahydrophillic semi-solid matrix.

Powders for constitution refers to powder blends containing the activeingredients and suitable diluents which can be suspended in water orjuices.

Suitable diluents are substances that usually make up the major portionof the composition or dosage form.

Suitable diluents include sugars such as lactose, sucrose, mannitol andsorbitol, starches derived from wheat, corn rice and potato, andcelluloses such as microcrystalline cellulose. The amount of diluent inthe composition can range from about 5 to about 95% by weight of thetotal composition, preferably from about 25 to about 75%, morepreferably from about 30 to about 60% by weight.

The term disintegrants refers to materials added to the composition tohelp it break apart (disintegrate) and release the medicaments. Suitabledisintegrants include starches, “cold water soluble” modified starchessuch as sodium carboxymethyl starch, natural and synthetic gums such aslocust bean, karaya, guar, tragacanth and agar, cellulose derivativessuch as methylcellulose and sodium carboxymethylcellulose,microcrystalline celluloses and cross-linked microcrystalline cellulosessuch as sodium croscarmellose, alginates such as alginic acid and sodiumalginate, clays such as bentonites, and effervescent mixtures. Theamount of disintegrant in the composition can range from about 2 toabout 20% by weight of the composition, more preferably from about 5 toabout 10% by weight.

Binders characterize substances that bind or “glue” powders together andmake them cohesive by forming granules, thus serving as the “adhesive”in the formulation. Binders add cohesive strength already available inthe diluent or bulking agent. Suitable binders include sugars such assucrose, starches derived from wheat, corn rice and potato; natural gumssuch as acacia, gelatin and tragacanth; derivatives of seaweed such asalginic acid, sodium alginate and ammonium calcium alginate; cellulosicmaterials such as methylcellulose and sodium carboxymethylcellulose andhydroxypropyl-methylcellulose; polyvinylpyrrolidone; and inorganics suchas magnesium aluminum silicate. The amount of binder in the compositioncan range from about 2 to about 20% by weight of the composition, morepreferably from about 3 to about 10% by weight, even more preferablyfrom about 3 to about 6% by weight.

Lubricant refers to a substance added to the dosage form to enable thetablet, granules, etc. after it has been compressed, to release from themold or die by reducing friction or wear. Suitable lubricants includemetallic stearates such as magnesium stearate, calcium stearate orpotassium stearate; stearic acid; high melting point waxes; and watersoluble lubricants such as sodium chloride, sodium benzoate, sodiumacetate, sodium oleate, polyethylene glycols and d,l-leucine. Lubricantsare usually added at the very last step before compression, since theymust be present on the surfaces of the granules and in between them andthe parts of the tablet press. The amount of lubricant in thecomposition can range from about 0.2 to about 5% by weight of thecomposition, preferably from about 0.5 to about 2%, more preferably fromabout 0.3 to about 1.5% by weight.

Glidents are materials that prevent caking and improve the flowcharacteristics of granulations, so that flow is smooth and uniform.Suitable glidents include silicon dioxide and talc. The amount ofglident in the composition can range from about 0.1% to about 5% byweight of the total composition, preferably from about 0.5 to about 2%by weight.

Coloring agents are excipients that provide coloration to thecomposition or the dosage form. Such excipients can include food gradedyes and food grade dyes adsorbed onto a suitable adsorbent such as clayor aluminum oxide. The amount of the coloring agent can vary from about0.1 to about 5% by weight of the composition, preferably from about 0.1to about 1%.

Techniques for the formulation and administration of the compounds ofthe present invention may be found in “Remington's PharmaceuticalSciences” Mack Publishing Co., Easton Pa., the entire contents of whichare hereby incorporated by reference. A suitable composition comprisingat least one compound of the invention may be a solution of the compoundin a suitable liquid pharmaceutical carrier or any other formulationsuch as tablets, pills, film tablets, coated tablets, dragees, capsules,powders and deposits, gels, syrups, slurries, suspensions, emulsions,and the like.

The term “treating and/or preventing” as used herein refers toreversing, alleviating, inhibiting the progress of, or preventing thedisorder or condition to which the term applies, or one or more symptomsof the disorder or condition. The term “treatment” as used herein refersto the act of treating and/or preventing as the term is defined above.Preferably, the treated or administered subject is a human subject andmore preferably a human subject in need of treatment.

The terms, “effective amount” or “therapeutically effective amount”means an amount sufficient to cause any observable or measurabledifference and preferably improvement in a subject's condition orindication, and preferably that condition or indication sought to betreated.

Accession numbers for murine anti-MIF antibodies are given below:

-   -   for the III.D.9 mAb: HB-12220 for the XIV.15.5 mAb: HB-12221.

Preferably, an “acute” condition, e.g. acute myocardial infarction, isdistinguished from a “chronic” condition, e.g., chronic congestive heartfailure.

When used herein, the term, “antibody” suitably includesantibody-derived fragment(s), Fab, Fab fragment(s), Fab₂, CDR-derivedregions, antibody-derived peptides, and/or single chain antibodies) asis known to those of ordinary skill in this art. Fab is preferred.

When used herein, the term, “cardiac dysfunction” may suitably includeone or more indications selected from the group including cardiacdysfunction, irregularity in myocardial activity, depression inmyocardial activity, burn-injury associated cardiac dysfunction, cardiacdysfunction following acute myocardial infarction, cardiodepression, anda combination thereof. These terms are understood by a physician ofordinary skill in this art.

Other embodiments of the present invention are given below.

-   A. A method for treating and/or preventing burn injury-associated    cardiodepression and/or cardiac dysfunction in a subject, which    includes:    -   administering to the subject an effective amount of at least one        anti-MIF antibody.-   B. A method for treating and/or preventing burn injury-associated    cardiac dysfunction in a subject, which includes:    -   administering to the subject an effective amount of at least one        anti-MIF antibody,    -   wherein the burn injury-associated cardiac dysfunction includes        irregularity in myocardial activity or depression in myocardial        activity or both.-   C. A method for treating and/or preventing burn injury-associated    cardiac dysfunction in a subject, which includes:    -   administering to the subject an effective amount of a        composition comprising an MIF inhibitor,    -   wherein the MIF inhibitor includes at least one anti-MIF        antibody.-   D. A method for treating and/or preventing burn injury-associated    cardiac dysfunction in a subject, which includes:    -   administering to the subject an effective amount of a        composition that includes at least one MIF inhibitor,    -   wherein the MIF inhibitor includes at least one anti-MIF        antibody, and wherein the antibody is a monoclonal antibody.-   E. A method for treating and/or preventing burn injury-associated    cardiac dysfunction in a subject, which includes:    -   administering to the subject an effective amount of a        composition that includes at least one MIF inhibitor,    -   wherein the MIF inhibitor includes at least one anti-MIF        monoclonal humanized antibody.-   F. A method for treating and/or preventing burn injury-associated    cardiac dysfunction in a subject, which includes:    -   administering to the subject an effective amount of a        composition that includes at least one MIF inhibitor, wherein        the composition is administered via at least one route selected        from the group including intramuscular injection,        intraperitoneal injection, subcutaneous injection, intravascular        injection, and a combination thereof.-   G. A pharmaceutical composition for the treatment and prevention of    burn injury-associated cardiac dysfunction in a subject, which    includes:    -   at least one anti-MIF antibody; and    -   at least one pharmaceutically acceptable carrier.-   H. A pharmaceutical composition for the treatment and prevention of    burn injury-associated cardiac dysfunction in a subject, which    includes:    -   at least one MIF inhibitor; and    -   at least one pharmaceutically acceptable carrier wherein said        MIF inhibitor includes at least one anti-MIF antibody.-   I. A method for treating and/or preventing cardiodepression and/or    cardiac dysfunction in a subject, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one anti-CD74 antibody.-   J. A method for treating and/or preventing cardiodepression and/or    cardiac dysfunction in a subject, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one anti-CD74 antibody,        wherein the cardiac dysfunction and/or cardiodepression includes        irregularity in myocardial activity or depression in myocardial        activity, or both.-   K. A method for treating and/or preventing cardiac dysfunction in a    subject, the method including:    -   administering to the subject an effective amount of at least one        anti-CD74 antibody, wherein the cardiac dysfunction includes at        least one burn injury-associated cardiac dysfunction.-   L. A method for treating and/or preventing cardiac dysfunction in a    subject, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one CD74 inhibitor, wherein        the CD74 inhibitor includes at least one anti-CD74 antibody.-   M. A method for treating and/or preventing cardiac dysfunction    and/or cardiodepression in a subject, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one CD74 inhibitor, wherein        the CD74 inhibitor includes at least one anti-CD74 monoclonal        antibody.-   N. A method for treating and/or preventing cardiac dysfunction in a    subject, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one CD74 inhibitor, wherein        the CD74 inhibitor includes at least one anti-CD74 monoclonal        humanized antibody.-   O. A method for treating and/or preventing cardiac dysfunction in a    subject, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one anti-CD74 antibody,        wherein the composition is administered via at least one route        selected from the group including intramuscular injection,        intraperitoneal injection, subcutaneous injection, intravascular        injection, and a combination thereof.-   P. A pharmaceutical composition for the treatment and prevention of    cardiac dysfunction and/or cardiodepression in a subject, which    includes:    -   at least one anti-CD74 antibody; and    -   at least one pharmaceutically acceptable carrier.-   Q. A pharmaceutical composition for the treatment and prevention of    cardiac dysfunction in a subject, which includes:    -   at least one CD74 inhibitor; and    -   at least one pharmaceutically acceptable carrier,    -   wherein the CD74 inhibitor includes at least one anti-CD74        antibody.-   R. A method for improving cardiac function in a subject following    acute myocardial infarction, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one anti-MIF antibody.-   S. A method for improving cardiac function in a subject following    acute myocardial infarction, the method including:    -   administering to the subject an effective amount of at least one        MIF inhibitor,    -   wherein the MIF inhibitor includes at least one anti-MIF        antibody.-   T. A method for improving cardiac function in a subject following    acute myocardial infarction, the method including:    -   administering to the subject an effective amount of at least one        MIF inhibitor,    -   wherein the MIF inhibitor includes at least one anti-MIF        monoclonal antibody.-   U. A method for improving cardiac function in a subject following    acute myocardial infarction, the method including:    -   administering to the subject an effective amount of at least one        MIF inhibitor,    -   wherein the MIF inhibitor includes at least one anti-MIF        monoclonal humanized antibody.-   V. A method for improving cardiac function in a subject following    acute myocardial infarction, the method including:    -   administering to the subject an effective amount of at least one        anti-MIF antibody and at least one anti-CD74 antibody.-   W. A method for improving cardiac function in a subject following    acute myocardial infarction, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one MIF inhibitor and at        least one CD74 inhibitor, wherein said CD74 inhibitor includes        at least one anti-CD74 antibody.-   X. A method for improving cardiac function in a subject following    acute myocardial infarction, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one MIF inhibitor and at        least one CD74 inhibitor, wherein the CD74 inhibitor includes at        least one anti-CD74 monoclonal antibody.-   Y. A method for improving cardiac function in a subject following    acute myocardial infarction, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one MIF inhibitor and at        least one CD74 inhibitor, and wherein the CD74 inhibitor        includes at least one anti-CD74 monoclonal humanized antibody.-   Z. A method for improving cardiac function in a subject following    acute myocardial infarction, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one anti-MIF antibody,    -   wherein the composition is administered via at least one route        selected from the group including intramuscular injection,        intraperitoneal injection, subcutaneous injection, intravascular        injection, and a combination thereof.-   AA. A pharmaceutical composition for the treatment and prevention of    cardiac dysfunction and/or cardiodepression in a subject, including:    -   at least one anti-MIF antibody; and    -   at least one pharmaceutically acceptable carrier.-   BB. A pharmaceutical composition for the treatment and prevention of    cardiac dysfunction and/or cardiodepression in a subject, including:    -   at least one anti-MIF antibody;    -   at least one anti-CD74 antibody; and    -   at least one pharmaceutically acceptable carrier.-   CC. A method for treating and/or preventing cardiac dysfunction in a    subject following acute myocardial infarction, the method including:    -   administering to the subject an effective amount of at least one        anti-TNFR antibody and at least one anti-MIF antibody.-   DD. A method for treating and/or preventing cardiac dysfunction in a    subject following acute myocardial infarction, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one TNFR inhibitor and at        least one MIF inhibitor,    -   wherein the TNFR inhibitor includes at least one anti-TNFR        antibody and wherein the MIF inhibitor includes at least one        anti-MIF antibody.-   EE. A method for treating and/or preventing cardiac dysfunction in a    subject following acute myocardial infarction, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one TNFR inhibitor and at        least one MIF inhibitor,    -   wherein the TNFR inhibitor includes at least one anti-TNFR        antibody and wherein the MIF inhibitor includes at least one        anti-MIF antibody,    -   wherein the anti-TNFR antibody includes at least one monoclonal        antibody.-   FF. A method for treating and/or preventing cardiac dysfunction in a    subject following acute myocardial infarction, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one TNFR inhibitor and at        least one MIF inhibitor,    -   wherein the TNFR inhibitor includes at least one anti-TNFR        antibody and wherein the MIF inhibitor includes at least one        anti-MIF antibody,    -   and wherein the anti-TNFR antibody includes at least one        monoclonal humanized antibody.-   GG. A method for treating and/or preventing cardiac dysfunction in a    subject following acute myocardial infarction, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one TNFR inhibitor and at        least one MIF inhibitor,    -   wherein the TNFR inhibitor includes at least one anti-TNFR        antibody and wherein the MIF inhibitor includes at least one        anti-MIF monoclonal antibody.-   HH. A method for treating and/or preventing cardiac dysfunction in a    subject following acute myocardial infarction, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one TNFR inhibitor and at        least one MIF inhibitor,    -   wherein the TNFR inhibitor includes at least one anti-TNFR        antibody and wherein the MIF inhibitor includes at least one        anti-MIF monoclonal humanized antibody.-   II. A method for treating and/or preventing cardiac dysfunction in a    subject following acute myocardial infarction, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one TNFR inhibitor and at        least one MIF inhibitor,    -   wherein the TNFR inhibitor includes at least one anti-TNFR        monoclonal humanized antibody and wherein the MIF inhibitor        includes at least one anti-MIF monoclonal humanized antibody.-   JJ. A pharmaceutical composition for the treatment and prevention of    cardiac dysfunction and/or cardiodepression in a subject, including:    -   at least one anti-TNFR antibody;    -   at least one anti-MIF antibody.-   KK. A pharmaceutical composition for the treatment and prevention of    cardiac dysfunction in a subject, including:    -   at least one TNFR inhibitor;    -   at least one MIF inhibitor; and    -   at least one pharmaceutically acceptable carrier;    -   wherein the TNFR inhibitor includes at least one anti-TNFR        antibody.-   LL. A pharmaceutical composition for the treatment and prevention of    cardiac dysfunction in a subject, including:    -   at least one TNFR inhibitor;    -   at least one MIF inhibitor; and    -   at least one pharmaceutically acceptable carrier;    -   wherein the MIF inhibitor includes at least one anti-MIF        antibody.-   MM. A pharmaceutical composition for the treatment and prevention of    cardiac dysfunction in a subject, including:    -   at least one TNFR inhibitor;    -   at least one MIF inhibitor; and    -   at least one pharmaceutically acceptable carrier;    -   wherein the TNFR inhibitor includes at least one anti-TNFR        antibody and    -   wherein the MIF inhibitor includes at least one anti-MIF        antibody.-   NN. A pharmaceutical composition for the treatment and prevention of    cardiac dysfunction in a subject, including:    -   at least one TNFR inhibitor;    -   at least one MIF inhibitor; and    -   at least one pharmaceutically acceptable carrier;    -   wherein the TNFR inhibitor includes at least one anti-TNFR        monoclonal antibody and wherein the MIF inhibitor includes at        least one anti-MIF monoclonal antibody.

OO. A pharmaceutical composition for the treatment and prevention ofcardiac dysfunction in a subject, including:

-   -   at least one TNFR inhibitor;    -   at least one MIF inhibitor; and    -   at least one pharmaceutically acceptable carrier;    -   wherein the TNFR inhibitor includes at least one anti-TNFR        monoclonal humanized antibody and wherein the MIF inhibitor        includes at least one anti-MIF monoclonal humanized antibody.

-   PP. A method for treating and/or preventing cardiac dysfunction in a    subject, the method including:    -   administering to the subject an effective amount of at least one        anti-MIF antibody.

-   QQ. A method for treating and/or preventing cardiac dysfunction in a    subject, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one anti-MIF antibody,    -   wherein the cardiac dysfunction includes at least one selected        from the group including irregularity in myocardial activity,        depression in myocardial activity, and a combination thereof.

-   RR. A method for treating and/or preventing cardiac dysfunction    and/or cardiodepression in a subject, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one MIF inhibitor,    -   wherein the MIF inhibitor includes at least one anti-MIF        antibody.

-   SS. A method for treating and/or preventing cardiac dysfunction in a    subject, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one MIF inhibitor,    -   wherein the MIF inhibitor includes at least one anti-MIF        monoclonal antibody.

-   TT. A method for treating and/or preventing cardiac dysfunction in a    subject, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one MIF inhibitor,    -   wherein the MIF inhibitor includes at least one anti-MIF        humanized antibody.

-   UU. A pharmaceutical composition, that includes:    -   a therapeutically effective amount of at least one anti-MIF        antibody; and    -   at least one pharmaceutically acceptable carrier.

-   VV. A pharmaceutical composition, which includes: at least one MIF    inhibitor; and    -   at least one pharmaceutically acceptable carrier,    -   wherein the MIF inhibitor includes at least one anti-MIF        antibody.

-   WW. A method for identifying an MIF inhibitor, the method including:    -   exposing at lease one myocyte to MIF;    -   determining at least one MIF-related myocyte activity;    -   exposing the myocyte to at least one MIF and at least one        candidate agent;    -   determining at least one MIF-related myocyte activity in the        presence of the candidate agent; and    -   determining whether the candidate agent affects the MIF-related        myocyte activity.

-   XX. A method for identifying an MIF inhibitor, the method including:    -   exposing at lease one myocyte to MIF;    -   determining at least one MIF-related myocyte activity;    -   exposing the myocyte to at least one MIF and at least one        candidate agent;    -   determining at least one MIF-related myocyte activity in the        presence of the candidate agent; and    -   determining whether the candidate agent affects the MIF-related        myocyte activity,    -   wherein the MIF-related myocyte activity is determined by        immunochemistry.

-   YY. A method for identifying an MIF inhibitor, the method including:    -   exposing at lease one myocyte to MIF;    -   determining at least one MIF-related myocyte activity;    -   exposing the myocyte to at least one MIF and at least one        candidate agent;    -   determining at least one MIF-related myocyte activity in the        presence of the candidate agent; and    -   determining whether the candidate agent affects the MIF-related        myocyte activity,    -   wherein the MIF-ralated myocyte activity is determined by        Langendorff assay.

-   ZZ A method for identifying an MIF inhibitor, the method including:    -   exposing at lease one myocyte to MIF;    -   determining at least one MIF-related myocyte activity;    -   exposing the myocyte to at least one MIF and at least one        candidate agent;    -   determining at least one MIF-related myocyte activity in the        presence of the candidate agent; and    -   determining whether the candidate agent affects the MIF-related        myocyte activity,    -   wherein the related myocyte activity is determined by        echocardiography.

-   AAA. A method for at least one selected from the group including    treating and/or preventing cardiac dysfunction in a subject in need    thereof, treating and/or preventing irregularity in myocardial    activity in a subject in need thereof, treating and/or preventing    depression in myocardial activity in a subject in need thereof,    treating and/or preventing burn-injury associated cardiac    dysfunction in a subject in need thereof, treating and/or preventing    cardiac dysfunction following acute myocardial infarction in a    subject in need thereof, treating and/or preventing cardiodepression    in a subject in need thereof, and a combination thereof, which    includes:    -   administering to the subject an effective amount of at least one        small molecule MIF inhibitor and/or salt thereof.

-   BBB. A method for at least one selected from the group including    treating and/or preventing cardiac dysfunction in a subject in need    thereof, treating and/or preventing irregularity in myocardial    activity in a subject in need thereof, treating and/or preventing    depression in myocardial activity in a subject in need thereof,    treating and/or preventing burn-injury associated cardiac    dysfunction in a subject in need thereof, treating and/or preventing    cardiac dysfunction following acute myocardial infarction in a    subject in need thereof, treating and/or preventing cardiodepression    in a subject in need thereof, and a combination thereof, which    includes:    -   administering to the subject an effective amount of a        composition that includes:    -   at least one small molecule MIF inhibitor and/or salt thereof;        and at least one pharmaceutically acceptable carrier.

-   CCC. A method for treating and/or preventing burn injury-associated    cardiodepression and/or cardiac dysfunction in a subject, which    includes:    -   administering to the subject an effective amount of at least one        small molecule MIF inhibitor and/or salt thereof.

-   DDD. A method for treating and/or preventing burn injury-associated    cardiac dysfunction in a subject, which includes:    -   administering to the subject an effective amount of at least one        small molecule MIF inhibitor and/or salt thereof,    -   wherein the burn injury-associated cardiac dysfunction includes        irregularity in myocardial activity or depression in myocardial        activity or both.

-   EEE. A method for treating and/or preventing burn injury-associated    cardiac dysfunction in a subject, which includes:    -   administering to the subject an effective amount of a        composition that includes at least one small molecule MIF        inhibitor and/or salt thereof.

-   FFF. A method for treating and/or preventing burn injury-associated    cardiac dysfunction in a subject, which includes:    -   administering to the subject an effective amount of a        composition that includes at least one small molecule MIF        inhibitor and/or salt thereof, wherein the composition is        administered via at least one route selected from the group        including intramuscular injection, intraperitoneal injection,        subcutaneous injection, intravascular injection, and a        combination thereof.

-   GGG. A pharmaceutical composition for the treatment and prevention    of burn injury-associated cardiac dysfunction in a subject, which    includes:    -   at least one small molecule MIF inhibitor and/or salt thereof;        and    -   at least one pharmaceutically acceptable carrier.

-   HHH. A method for improving cardiac function in a subject following    acute myocardial infarction, the method including:    -   administering to the subject an effective amount of at least one        small molecule MIF inhibitor and/or salt thereof.

-   III. A method for improving cardiac function in a subject following    acute myocardial infarction, the method including:    -   administering to the subject an effective amount of a        composition that includes at least one small molecule MIF        inhibitor and/or salt thereof,    -   wherein the composition is administered via at least one route        selected from the group including intramuscular injection,        intraperitoneal injection, subcutaneous injection, intravascular        injection, and a combination thereof.

-   JJJ. A method for treating and/or preventing cardiac dysfunction in    a subject following acute myocardial infarction, the method    including:    -   administering to the subject an effective amount of a        composition that includes at least one small molecule MIF        inhibitor and/or salt thereof.

-   KKK. A pharmaceutical composition effective for at least one    selected from the group including treating and/or preventing cardiac    dysfunction in a subject in need thereof, treating and/or preventing    irregularity in myocardial activity in a subject in need thereof,    treating and/or preventing depression in myocardial activity in a    subject in need thereof, treating and/or preventing burn-injury    associated cardiac dysfunction in a subject in need thereof,    treating and/or preventing cardiac dysfunction following acute    myocardial infarction in a subject in need thereof, treating and/or    preventing cardiodepression in a subject in need thereof, and a    combination thereof, which includes an effective amount of a    combination that includes:    -   at least one anti-TNFR antibody;    -   at least one anti-MIF antibody; and    -   at least one pharmaceutically acceptable carrier.

-   LLL. A pharmaceutical composition effective for at least one    selected from the group including treating and/or preventing cardiac    dysfunction in a subject in need thereof, treating and/or preventing    irregularity in myocardial activity in a subject in need thereof,    treating and/or preventing depression in myocardial activity in a    subject in need thereof, treating and/or preventing burn-injury    associated cardiac dysfunction in a subject in need thereof,    treating and/or preventing cardiac dysfunction following acute    myocardial infarction in a subject in need thereof, treating and/or    preventing cardiodepression in a subject in need thereof, and a    combination thereof, which includes an effective amount of a    combination that includes:    -   at least one anti-CD-74 antibody;    -   at least one anti-MIF antibody; and    -   at least one pharmaceutically acceptable carrier.

-   MMM. A method for at least one selected from the group including    treating and/or preventing cardiac dysfunction in a subject in need    thereof, treating and/or preventing irregularity in myocardial    activity in a subject in need thereof, treating and/or preventing    depression in myocardial activity in a subject in need thereof,    treating and/or preventing burn-injury associated cardiac    dysfunction in a subject in need thereof, treating and/or preventing    cardiac dysfunction following acute myocardial infarction in a    subject in need thereof, treating and/or preventing cardiodepression    in a subject in need thereof, and a combination thereof, which    includes administering to the subject an effective amount of a    combination that includes:    -   at least one anti-TNFR antibody;    -   at least one anti-MIF antibody; and    -   at least one pharmaceutically acceptable carrier.

-   NNN. A method for at least one selected from the group including    treating and/or preventing cardiac dysfunction in a subject in need    thereof, treating and/or preventing irregularity in myocardial    activity in a subject in need thereof, treating and/or preventing    depression in myocardial activity in a subject in need thereof,    treating and/or preventing burn-injury associated cardiac    dysfunction in a subject in need thereof, treating and/or preventing    cardiac dysfunction following acute myocardial infarction in a    subject in need thereof, treating and/or preventing cardiodepression    in a subject in need thereof, and a combination thereof, which    includes administering to the subject an effective amount of a    combination that includes:    -   at least one anti-CD-74 antibody;    -   at least one anti-MIF antibody; and    -   at least one pharmaceutically acceptable carrier.

-   OOO. Another embodiment of the invention relates to administering an    effective amount of one or more of the soluble MIF receptor and/or    MIF receptor antagonist (optionally in a pharmaceutically acceptable    carrier) to a subject in need thereof for at least one selected from    the group including treating and/or preventing cardiac dysfunction    in a subject in need thereof, treating and/or preventing    irregularity in myocardial activity in a subject in need thereof,    treating and/or preventing depression in myocardial activity in a    subject in need thereof, treating and/or preventing burn-injury    associated cardiac dysfunction in a subject in need thereof,    treating and/or preventing cardiac dysfunction following acute    myocardial infarction in a subject in need thereof, treating and/or    preventing cardiodepression in a subject in need thereof, and a    combination thereof.

-   PPP. Another embodiment of the invention relates to administering an    effective amount of one or more of the following, in any    combination:    -   small molecule MIF inhibitor;    -   soluble MIF receptor;    -   MIF receptor antagonist;    -   anti-CD74 antibody;    -   anti-MIF antibody;    -   anti-TNFR antibody; and    -   optionally, a pharmaceutically acceptable carrier    -   to a subject in need thereof for at least one selected from the        group including treating and/or preventing cardiac dysfunction        in a subject in need thereof, treating and/or preventing        irregularity in myocardial activity in a subject in need        thereof, treating and/or preventing depression in myocardial        activity in a subject in need thereof, treating and/or        preventing burn-injury associated cardiac dysfunction in a        subject in need thereof, treating and/or preventing cardiac        dysfunction following acute myocardial infarction in a subject        in need thereof, treating and/or preventing cardiodepression in        a subject in need thereof, and a combination thereof.

-   QQQ. Another embodiment of the invention relates to a composition,    that includes an effective amount of one or more of the following,    in any combination:    -   small molecule MIF inhibitor;    -   soluble MIF receptor;    -   MIF receptor antagonist;    -   anti-CD74 antibody;    -   anti-MIF antibody;    -   anti-TNFR antibody; and    -   optionally, a pharmaceutically acceptable carrier    -   wherein the composition is effective for at least one selected        from the group including treating and/or preventing cardiac        dysfunction in a subject in need thereof, treating and/or        preventing irregularity in myocardial activity in a subject in        need thereof, treating and/or preventing depression in        myocardial activity in a subject in need thereof, treating        and/or preventing burn-injury associated cardiac dysfunction in        a subject in need thereof, treating and/or preventing cardiac        dysfunction following acute myocardial infarction in a subject        in need thereof, treating and/or preventing cardiodepression in        a subject in need thereof, and a combination thereof.

-   RRR. Another embodiment of the invention relates to administering an    effective amount of anti-TNFR antibody; and    -   optionally, a pharmaceutically acceptable carrier to a subject        in need thereof for at least one selected from the group        including treating and/or preventing cardiac dysfunction in a        subject in need thereof, treating and/or preventing irregularity        in myocardial activity in a subject in need thereof, treating        and/or preventing depression in myocardial activity in a subject        in need thereof, treating and/or preventing burn-injury        associated cardiac dysfunction in a subject in need thereof,        treating and/or preventing cardiac dysfunction following acute        myocardial infarction in a subject in need thereof, treating        and/or preventing cardiodepression in a subject in need thereof,        and a combination thereof.

-   SSS. Another embodiment of the invention relates to a composition    that includes an effective amount of anti-TNFR antibody; and    -   optionally, a pharmaceutically acceptable carrier    -   wherein the composition is effective for at least one selected        from the group including treating and/or preventing cardiac        dysfunction in a subject in need thereof, treating and/or        preventing irregularity in myocardial activity in a subject in        need thereof, treating and/or preventing depression in        myocardial activity in a subject in need thereof, treating        and/or preventing burn-injury associated cardiac dysfunction in        a subject in need thereof, treating and/or preventing cardiac        dysfunction following acute myocardial infarction in a subject        in need thereof, treating and/or preventing cardiodepression in        a subject in need thereof, and a combination thereof.

EXAMPLES

Having generally described this invention, a further understanding canbe obtained by certain specific examples, which are provided herein forpurposes of illustration only, and are not intended to be limitingunless otherwise specified.

Example 1

Antibodies and cytokines. Goat anti-hMIF IgG and rhMIF (R&D Systems,Minneapolis, NIN) were reconstituted in PBS and 0.1% BSA in PBSrespectively, aliquoted, and stored at −20° C. until use. Rabbitanti-goat IgG-HRP (BioRad Corp., Hercules, Calif.) stored at 4° C. untiluse.

Animals and Experimental Design. C57BL/6J and C3H/HeJ mice were obtainedat 6-10 weeks of age (Jackson Labs, Bar Harbor, Me.). AdultSprague-Dawley rats (Harlan Laboratories, Houston, Tex.) weighing325-360 g were used in this study. Commercial chow and tap water weremade available ad libitum. All animal protocols were reviewed andapproved by the University of Texas Southwestern Medical CenterInstitutional Animal Care Advisory Committee and were in compliance withthe rules governing animal use as published by the NIH. C57BL/6J micewere injected i.p. with 4 mg/kg E. coli 0111:B4 LPS (Sigma-AldrichCorp., St. Louis, Mo.) and sacrificed post injection at time pointsindicated in the text by CO₂ asphyxiation and subsequent cervicaldislocation. Uninjected mice were used as controls. Two anti-MIFantibodies (III.D.9 and XIV.15.5, Rockland Immunochemicals, Inc.,Gilbertsville, Pa.) and their isotype control (HB-49, RocklandImmunochemicals, Inc., Gilbertsville, Pa.) were injected (100 μg in 200μl PBS) i.p. 90 m before the LPS challenge in the echocardiogramstudies. Whole hearts were removed and snap frozen in liquid nitrogenand stored at −80° C. or fixed in 10% neutral-buffered formalin for 24 hand placed in 70% ethanol for immunohistochemistry.

Example 2

Protein Extraction and Western Blotting. Hearts were thawed andhomogenized on ice in Tris-Buffered Saline (TBS, 50 mM Tris, 150 mMNaCl, pH 7.5) containing 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, 2 mMEDTA, and 1 mM PMSF. Lysate concentration was quantified using theBio-Rad Protein Assay (Hercules, Calif.). Protein (20 μg) was diluted1:1 with Laemmli sample buffer (Bio-Rad, Hercules, Calif.) and resolvedon an 18% SDS polyacrylamide gels under reducing conditions. The gel wastransferred to PVDF membranes (NEN, Boston, Mass.) using a semi-drytransfer apparatus (Bio-Rad, Hercules, Calif.) at 15 V for 15 m.Membranes were blocked with TBS/0.1% Tween-20 (TBS-T) with 0.5% nonfatdry milk for 30 in and incubated with goat anti-hMIF IgG (1:750) inTBS/0.1% Tween-20/5% nonfat milk overnight at 4° C. The membranes werewashed 3 times for 10 m in TBS-T, incubated with rabbit anti-goatIgG-HRP (1:1000) for 1 h at RT, and washed 4 times for 10 m with TBS-T.The membranes were exposed to 5 ml of a mixture of luminol plus hydrogenperoxide under alkaline conditions (SuperSignal West Pico, Pierce,Rockford, Ill.) for 5 min and the resulting chemiluminescent reactionwas detected by Kodak X-OMAT AR Film (Eastman Kodak Co., Rochester,N.Y.).

Example 3

RNA Extraction, probe preparation and Northern Blotting. Total RNA wasextracted with Trizol (Invitrogen, Carlsbad, Calif.) from hearts thawedon ice according to the manufacturer's protocol and quantified byspectrophotometry. A MIF specific Northern probe was prepared byisolating DNA (DNeasy Tissue Kit, Qiagen, Valencia, Calif.) from the MIFplasmid (Research Genetics, Huntsville, Ala.) and subsequently cuttingit with ECOR1 and NOT I restriction enzymes (Fisher Scientific,Pittsburgh, Pa.). The resultant DNA was resolved on a 1.2% agarose gel,purified (GenElute Agarose Spin Columns, Supelco, Bellefonte, Pa.),labeled with 5 μl ³²P-dCTP (3000 Ci/mmol)(PerkinElmer, Boston, Mass.)using Ready-To-Go Labeling Beads (Amersham Pharmacia, Piscatany, N.J.),and purified in ProbeQuant Microcolumns (Amersham Pharmacia, Piscatany,N.J.) according to manufacturer's protocols.

mRNA (10 μg) was resolved on 1.2% agarose gels at 100 V for 1 h andtransferred to Hybond-N+ membranes (Amersham Pharmacia, Buckingham,England) at 100 V for 1 h on a transfer electrophoresis unit (TransPhorPowerLid, Hoefer Scientific Instruments, San Francisco). RNA was linkedto the membranes for 2 m using a GS Gene Linker (Bio-Rad, Hercules,Calif.). Membranes were prehybridized in Perfect-Hyb Plus (Sigma, St.Louis, Mo.) for 4-5 h at 42° C. and then incubated with ³²P labeled MIFDNA probe at 42° C. overnight. The membranes were washed twice for 30 min 2×SSC/0.1% SDS at 46° C., and washed twice for 30 m in 0.2×SSC/0.1%SDS at 46° C., and detected by Kodak X-OMAT AR Film (Eastman Kodak Co.,Rochester, N.Y.). The same membranes were then probed with radiolabeledβ-actin to ensure equal loading of protein.

Example 4

Immunohistochemistry. Tissue was fixed in neutral buffered formalin andprocessed to paraffin and subsequently immunostained at RT on a BioTekSolutions Techmate™ 1000 automated immunostainer (Ventana MedicalSystems, Tucson, Ariz.) using the Ultra-streptavidin biotin system withhorseradish peroxidase and diaminobenzidine (DAB) chromogen (SignetLaboratories, Dedham, M A). Optimum primary antibody dilutions werepredetermined using known positive control tissues (rat post-LPSchallenge). Paraffin sections were cut at 3 μm on a rotary microtome,mounted on positively charged glass slides (POP100 capillary gap slides,Ventana Medical Systems, Tucson, Ariz.) and air-dried overnight.Sections were then deparaffinized in xylene and ethanol, quenched withfresh 3% hydrogen peroxide for 10 m to inhibit endogenous tissueperoxidase activity, and rinsed with deionized water. Sections wereincubated in unlabeled blocking serum for 15 m to block nonspecificbinding of the secondary antibody and then incubated for 25 m witheither rabbit anti-MIF (1:400, Torrey Pines BioLabs, Inc., Houston,Tex.) diluted in 1% citrate buffer (BioPath, Oklahoma City, Okla.), orwith buffer alone as a negative reagent control. Following washes inbuffer, sections were incubated for 25 in with a biotinylated polyvalentsecondary antibody solution (containing goat anti-rabbitimmunoglobulins). Next, sections were washed with buffer, incubated inhorseradish peroxidase-conjugated streptavidin-biotin complex for 15 in,washed again in buffer, and then incubated with 2 changes, 5 in each, ofa freshly prepared mixture of DAB and H₂O₂ in buffer, followed bywashing in buffer and then water. Sections were then counterstained withhematoxylin, dehydrated in a graded series of ethanol and xylene, andcoverslipped. Slides were reviewed by light microscopy and positivereactions with DAB were identified as a dark brown reaction product.

Example 5

Determination of cardiac function in response to rMIF. C57BL/6J andC₃H/HeJ mice were used in the Langendorff assays. Briefly, 200 U heparinsulfate was given i.p., the mice sacrificed 20 m later, and the heartimmediately removed and placed on ice in Krebs-Hanseleit Buffer (2 mMNaHCO₃, 118 mM NaCl, 4.7 mM KCL, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.5 mMCaCl₂, 11.1 mM glucose, pH 7.4, which was prepared fresh withdemineralized, deionized water and bubbled with 95% O₂ and 5% CO₂ (PO₂590 mmHg, pCO₂ 38 mmHg)). The aorta was cannulated with PE50 tubing, theheart perfused in a retrograde manner through the aortic root withprefiltered, oxygenated Krebs-Hanseleit Buffer at a constant flow rateof 1.5 ml/m (T 37° C.) and a recirculating volume of 100 ml. The heartwas placed in a water-jacketed chamber to maintain constant temperatureand humidity. PE60 intratnedic polyethylene tubing was connected to aStatham pressure transducer inserted into the left ventricle (LV) tomeasure LV pressure. Temperature was monitored using a 27 G thertnistorneedle inserted into the LV muscle. After instrumentation, hearts wereallowed to stabilize for 10 m and hearts that failed to achieve a stablepressure or developed persistent arrhythmias during this time wereexcluded from the study. Following stabilization, LV pressure and itsfirst derivative (dP/dt), heart rate, and coronary perfusion weremeasured simultaneously with a multichannel Grass 7D polygraph (GrassInstruments, Quincy, M A). Cardiac function for all hearts wasdetermined by plotting peak systolic LV pressure and ±dP/dt,Ia, valuesagainst changes in coronary flow rate. Hearts were perfused with orwithout 20 ng/ml RMIF added to the perfusate.

Example 6

Determination of cardiac dysfunction by echocardiography.Echocardiograms to assess systolic function were performed using M-modemeasurements. Mice were anesthestized with 5% isofluorane with 2.5 L/mO₂ for 20 seconds (until unconscience) followed by 2% isofluorane and O₂for an average of 12-15 m. Hair was removed from the thorax and upperabdomen using Nair® hair remover after sitting for 3 m using gauze.Echocardiography measurements were obtained on anesthetized miceapproximately 5-8 m after induction to allow any transient cardiacdepression to pass. These transient, minimal changes in cardiac functiondetected by echocardiography have been reported using inhaledisofluorane, although FS (%) has been reported to be stable. Cardiacechocardiography was performed using a Hewlett-Packard Sonos 5500(Agilent Technologies; Edmonton, Alberta, Canada) with a frame rate of300-500 frames/s in a random and blinded manner. A 12 MHz lineartransducer was placed on the left hemithorax interfaced with a layer ofUS transmission gel (Aquasonic 100, Parker Laboratories; Fairfield,N.J.). The two dimensional parastemal short-axis imaging plane guided LVM-mode tracings close to the papillary muscle level. Depth was set at aminimum of 2 cm with a sweep speed of 150 m/s. Tracings were printed ona Sony color printer (UP-5200, Sony).

Example 7

M-mode measurements. Data represented the average of at least twoseparate scans, each of which represent the average of at least threeselected beats. End diastole was defined as the maximal LV diastolicdimension, and end systole was defined as the peak of posterior wallmotion. Systolic function was calculated from LV dimensions asfractional shortening (FS) as follows: FS (%): LVED−LVES/LVED×100, asshown in FIG. 5A.

Example 8

Statistical Analysis. Northern and Western data are expressed asmean±standard error and statistically analyzed using a One Way Analysisof Variance. Determination of significance between experimental andcontrol groups was performed using the Tukey method (p≦0.05). Cardiacfunction determined by the Langendorff preparation is expressed asmean±standard error and separate analyses were performed for each ofLVP, +dP/dt_(max), and −dP/dt_(max), as a function of treatment groupand coronary flow rate using a Repeated Measures Analysis of Variance. Amultiple comparison procedure employing the Bonferroni method was usedto determine significant differences between groups (p<0.05). Cardiacfunction determined by echocardiogram is expressed by fractionalshortening % (LVED−LVES/LVEP×100.)±standard deviation and analyzed usinga One Way Repeated Measures Analysis of Variance. Additional comparisonswere performed using the Tukey Test to determine significant differencesbetween specific groups (p<0.05). All statistical analyses wereperformed using SigmaStat 2.03 (SPSS Inc., Chicago, Ill.) and MicrosoftExcel (Microsoft Corp., Seattle, Wash.).

Example 9

MIF protein is constituitively expressed by cardiac myocytes in vivo andis released in response to LPS Challenge. Both immunochemistry andWestern analysis performed on cardiac tissue documented the presence ofMIF in cardiac cells, including ventricular and atrial myocytes, underbaseline control conditions (FIGS. 1 and 2). Following endotoxinchallenge, both immunochemistry and immunoblot analysis document asignificant decrease in cardiac tissue MIF following endotoxin. Thisdecrease was most profound (75% decrease) at 12 h, but returned to nearbaseline control levels by 24 h. This expression pattern in the heart issimilar to that witnessed in the liver and spleen (FIG. 2), andconsistent with the hypothesis that MIF is released from preformedstores within tissue following LPS challenge. The release of MIF fromtissue is evident at 4 hours on immunoblot (FIG. 1) correlates with theincrease in serum levels following endotoxin exposure (Table 1). TABLE 1Serum Levels of MIF Following a 4 mg/kg Endotoxin Challenge. 3mice/group; perform statistics (One Way ANOVA) Baseline 4 hours 8 hours12 hours 24 hours 48 hours 79.1 ± 4.6 90.9 ± 8.7 118.1 ± 5.6* 81.4 ± 5.870.1 ± 5.1 69.9 ± 9.0

Example 10

Myocardial MIF mRNA Expression Following Endotoxin Challenge. Northernanalysis of RNA obtained from the hearts of either control mice or fromLPS challenged mice at given time points indicates that MIF mRNA isconstitutively expressed in control mice, and that after LPS challenge,no significant change in MIF mRNA concentration is detectable in wholeheart preparations (FIG. 3).

Example 11

MIF induces systolic and diastolic cardiac dysfunction. To determine ifMIF directly influences cardiac function, spontaneously beating normalmouse hearts (Langendorff preparation) were perfused with recombinantMIF (rMIF) at a concentration of 20 ng/ml, approximating that documentedin the serum of humans with septic shock (20). Responses to MIF weredetermined in hearts from both C57BL/6J mice, and C3H/HeJ mice. C3H/HeJmice are resistant to endotoxin (41-43), therefore controlling for thepossibility that any depression observed might be due to trace endotoxinin the perfusate. Table 2 illustrates the responses of both mousestrains to retrograde aortic perfusion at 1.5 ml/m with controlperfusate or perfusate containing 20 ng/ml recombinant MIF. Perfusionwith MIF led to a significant decrease in LVP, +dP/dt_(max), and −dP/dtmax in both mouse strains. FIG. 4 illustrates the effect of MIF over arange of coronary flow rates. There is a step-wise increase incontractile performance in all hearts regardless of experimental groupassignment. Comparison of the MIF exposed hearts with control heartsrevealed a downward shift in the function curves, indicating significantsystolic and diastolic depression in response to 20 ng/ml rMIF (p<0.05).The effect of MIF was statistically identical in both endotoxinsensitive (C57BL/6J) and endotoxin resistant (C3H/HeJ) strains.Likewise, there were no differences in LVP, +dP/dt_(max), and−dP/dt_(max), between the C57BL/6J and C3H/HeJ study hearts perfusedwith rMIF. TABLE 2 Mean peak cardiac function before and after treatmentwith rMIF in a Landendorf preparation in C57BL/6J mice and LPS-resistantC3H/HeJ mice. +dP/dt_(max) −dP/dt_(max) LVP (mm Hg) (mm Hg/see) (mmHg/sec) C57BL/6J 92.9 ± 2.3 2180 ± 60  1856 ± 65  Control (n = 10)C57BL/6J  77.9 ± 5.1* 1920 ± 89*  1545 ± 122* rMIF (n = 10) C3H/HeJ 93.7± 3.5 2250 ± 42  1809 ± 84  Control (n = 7) C3H/HeJ  75.2 ± 5.5* 1800 ±106* 1343 ± 138* rMIF (n = 7)*p < 0.05.

Example 12

Anti-MIF antibodies improve LPS-induced cardiac depression in vivo. Todetermine the influence of MIF in the pathogenesis of cardiacdysfunction in vivo, serial echocardiography (M-mode) was performed onLPS challenged mice which had been pre-treated (90 minutes prior) witheither anti-MIF monoclonal antibodies, an isotype control antibody, orno treatment (FIG. 5). At four hours post-LPS challenge, the fractionalshortening % (FS %) of all LPS challenged mice were similarly depressed(50% reduction in FS %), irrespective of group assignment. Eight hourspost-LPS challenge, however, mice injected with either anti-MIFmonoclonal antibody demonstrated statistically significant recovery ofFS % compared to LPS challenged groups receiving either no treatment orisotype antibody control (FIG. 5). This enhanced recovery of functioncontinued at 12, 24, and 48 h. At 48 hours following challenge, anti-MIFtreated groups had near total restoration of FS %, whereas LPSchallenged groups remained profoundly depressed. Throughout the 48hours, the FS % of sham mice did not significantly change, indicatingthat cardiac function was unaffected by anesthesia or the testingregimen itself. Additionally, at all time points, the mice injected withisotypic antibody controls were identical to animals challenged withLPS, indicating specificity of the anti-MIF antibody effects.

Example 13

Materials and Methods

Antibodies and cytokines. A polyclonal rabbit anti-rat MIF IgG (TorreyPines BioLabs, Inc., Houston, Tex.) was used for western immunoblot andimmunohistochemistry. This antibody has previously been shown to crossreact with murine MIF and was prepared as previously described (23). Apolyclonal goat anti-rabbit IgG-HRP (BioRad Corp., Hercules, Calif.) wasused as a secondary antibody for western immunoblots and was stored at4° C. Two monoclonal mouse anti-mouse (and human) MIF IgG1 antibodies(XIV.15.5 and III.D.9, gift of Cytokine PharmaSciences, Inc.) and amonoclonal mouse IgG1 isotype control antibody (HB-49, gift fromCytokine PharmaSciences, Inc.) were used in the echocardiographicstudies. In vivo neutralization of MIF activity by both the XIV.15.5 andIII.D.9 clones have been previously demonstrated.

Animals, Experimental Design, and Burn Injury. Male C57BL/6J mice ages6-10 weeks (Jackson Labs, Bar Harbor, Me.) were maintained in a specificpathogen free environment. Commercial chow and tap water were madeavailable ad libitum. All animal protocols were reviewed and approved bythe University of Texas Southwestern Medical Center Institutional AnimalCare Advisory Committee and were in compliance with the rules governinganimal use as published by the NIH. Mice were subjected to a 40% TBSAburn injury. Briefly, mice were anesthetized with isoflourane (1-2%)with 2.5 L/minute oxygen to effect. Hair was then removed from theirback and sides using a surgical prep blade and 70% ethanol. Brass probesheated to 100° C. in boiling water were then applied in pairs (total of8 probe surface areas) on the animal's side and back for 5 seconds.Alternatively, sham mice received anesthesia and were shaven but notgiven the burn injury. Intraperitoneal injection of Lactated Ringer'swith Buprenex (2 cc LR+0.2 cc Buprenex (=0.05 mg/kg)) was given afterthe burn injury after the anesthesia was removed (with oxygencontinued). Mice were then placed in individual cages under a heat lampfor approximately 1 hour and on a heating pad for the duration of thestudy and monitored closely. Mice were sacrificed at time pointsindicated in the figures by CO₂ asphyxiation followed by cervicaldislocation. Monoclonal anti-MIF antibodies (III.D.9 and XIV.15.5) or anisotypic control (HB-49) were injected (100 μg in 200 μl PBS)intraperitoneally 90 minutes prior to burn injury in the echocardiogramstudies. Whole hearts were removed, snap frozen in liquid nitrogen, andstored at −80° C. In parallel experiments, hearts were fixed in 10%neutral-buffered formalin for 24 hours and were then placed in 70%ethanol until they were processed for immunohistochemistry. Whole bloodwas collected by retro-orbital bleeding and serum collected and stored.

Protein Extraction and Western Blotting. Hearts stored at −80° C. werehomogenized on ice in lysate buffer (10 mM HEPES, 2 mM EDTA, 0.1% Chaps,pH 7.4 with one Complete Mini-EDTA-Free Protease inhibitor cocktailtablet per 10 ml buffer, Roche Diagnostics, Mannheim, Germany). Proteinconcentration was quantified using the Bio-Rad Protein Assay (Hercules,Calif.). Fifty μg of total protein (lysate) diluted in Laemmeli samplebuffer (Bio-Rad) in a 1:1 ratio to a final volume of 10 μl was thenresolved on a 12% SDS polyacrylamide gel under reducing conditions.Prestained SDS-PAGE standards (Kaleidoscope Broad range, Bio-RadLaboratories, Inc., Hercules, Calif.) were run with each gel in order todetermine the approximate M.W. of detected bands. The gel wastransferred to a PVDF membrane (NEN, Boston, Mass.) using a minitransblot transfer apparatus (Bio-Rad, Hercules, Calif.) at 100 V for 70minutes and cooled with ice packs. The membrane was re-wet withmethanol, washed a minimum of 3 times with 100+ ml water, and blocked(5% nonfat dry milk (Bio-Rad)/TBS/0.1% Tween-20 (TBS-T) overnight at 4°C. The membrane was then incubated with the primary rabbit anti-MIF(1:1250 dilution) for 2 hours at room temperature in 5% milk/TBS-T andwashed once for 15 minutes in TBS-T, followed by five washes (5 minuteseach) in TBS-T. It was then incubated for 1 hour with a HRP conjugatedgoat anti-rabbit antibody in TBS-T (1:5000) at room temperature, washedtwice for 15 minutes, followed by five additional washes (5 minuteseach) in TBS-T. To develop, 5 ml of ECL reagent (SuperSignal West Pico,Pierce, Rockford, Ill.) was placed on the PVDF membranes for 5 minutes,and the resulting chemiluminescent reaction was detected by Kodak X-OMATAR Film (Eastman Kodak Co., Rochester, N.Y.).

The quantification of the single band density with the approximatemolecular weight of MIF (12.5 kD) was determined using Quantity Onesoftware (Bio-Rad, Hercules, Calif., Ver. 4.4.0, Build 36) followingconversion of radiographic film to TIFF files (8 bit grayscale) using aScanjet 3400c (Hewlett Packard, Palo Alto, Calif.) and reported inarbitrary units (A.U.)/mm².

Immunohistochemistry. Tissue was fixed in neutral buffered formalin,processed to paraffin, and subsequently immunostained at roomtemperature on a BioTek Solutions Techmate™1000 automated immunostainer(Ventana Medical Systems, Tucson, Ariz.) using the Ultra-streptavidinbiotin system with horseradish peroxidase and diaminobenzidine (DAB)chromogen (Signet Laboratories, Dedham, Mass.). Optimum primary antibodyconcentrations were predetermined using known positive control tissues(LPS challenged rat). Paraffin sections were cut at 3 μm on a rotarymicrotome, mounted on positively charged glass slides (POP100 capillarygap slides, Ventana Medical Systems, Tucson, Ariz.), and air-driedovernight. Sections were then deparaffinized in xylene and ethanol,quenched with fresh 3% hydrogen peroxide for 10 minutes to inhibitendogenous tissue peroxidase activity, and rinsed with de-ionized water.Sections were incubated in unlabeled blocking serum for 15 minutes toblock nonspecific binding of the secondary antibody and then incubatedfor 25 minutes with either the polyclonal rabbit anti-rat MIF IgG(1:400, Torrey Pines BioLabs, Inc., Houston, Tex.) diluted in 1% citratebuffer (BioPath, Oklahoma City, Okla.) or with buffer alone as anegative reagent control. A negative reagent control was run for eachtime point and for each organ. Following washes in buffer, sections wereincubated for 25 minutes with a biotinylated polyvalent secondaryantibody solution (containing goat anti-rabbit immunoglobulins). Next,sections were washed with buffer, incubated in horseradishperoxidase-conjugated streptavidin-biotin complex for 15 minutes, washedagain in buffer, and then incubated with 2 changes, 5 minutes each, of afreshly prepared mixture of DAB and H₂O₂ in buffer, followed by washingin buffer and then water. Sections were then counterstained withhematoxylin, dehydrated in a graded series of ethanol and xylene, andcoverslipped. Slides were reviewed by light microscopy, and positivereactions with DAB were identified as a dark brown reaction product.

Determination of serum MIF levels. Sera from six mice were assayed formouse MIF using the Chemikine” Rat/Mouse macrophage inhibitory factor(MIF) EIA kit (Chemicon International, Inc., Temecula, C A) according tothe manufacture's instructions. Briefly, 5 μl of standards, samples, orreaction buffer (blank) were added to each well in triplicate. Next, 100μl of diluted MIF-HRP antibody conjugate was added to each well andallowed to incubate for 2 hours at room temperature. Wells were thenwashed five times, and 100 μl of 3, 3′, 5, 5′-tetramethylbenzidene (TMB)substrate was added and allowed to incubate in the dark for 30 minutesat room temperature. The stop reagent was added to each well, gentlymixed, and the ELISA was read on an ELISA plate reader (EL 312eMicroplate Reader, Bio-Tek Instruments, Winooski, Vt.) at 450 nm (630 nmbackground) within 30 minutes of completion of the assay.

Multiplex cytokine detection by Luminex. Plasma inflammatory cytokine(IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFN-γ, TNF-α, and GM-CSF)concentrations were determined using the Mouse Cytokine Ten-PlexAntibody Bead Kit (Biosource International, Inc., Camarillo, Calif.) ona Luminex xMAP” system (Luminex Corp., Austin, Tex.) according to themanufacturer's instructions. The plate was loaded onto the Luminex XYP™platform, the instrument set to remove 50 μl, and the total event set toequal the 100 per bead set. At least 100 events (most >200) for eachcytokine were collected in each sample in order to determinestatistically significant results. Data was collected using the Luminex™Data Collector Software (Luminex Corp., Austin, Tex.). Theconcentrations of the lot specific reconstituted standards used in eachrun were entered into the software and the analyte concentrations forunknown samples were then extrapolated from the cytokine specificstandard curve using MasterPlex™ QT software (Version 1.2.8.58, MiraiBio, Inc., Alameda, Calif.). Final concentrations were multiplied by 2in order to account for the initial dilution factor. No samples weredetected that were higher than the standards curves for any cytokine.

Total RNA isolation, MIF and β-actin probe preparation, and northernblotting. Hearts stored at −80° C. were placed in a liquid nitrogenfilled pestle and ground to a fine powder with a mortar. Each powderedheart was then immediately placed in 2 ml Trizol (Invitrogen, Carlsbad,Calif.) and total RNA was isolated according to the manufacturer'sprotocols and quantified by spectrophotometry. An MIF specific Northernprobe was prepared from an MIF containing plasmid (Image Clone I.D.634910, Research Genetics, Huntsville, Ala.) isolated using Genelute HPPlasmid MidiPrep kit (Sigma, St. Louis, Mo.). The fragment was preparedby an EcoR1 and Not1 digestion (Fisher Scientific, Pittsburgh, Pa.) andgel purified and isolated on a 1.2% agarose gel using GenElute AgaroseSpin Columns (Supelco, Bellefonte, Pa.). The β-actin probe DNA fragmentwas purchased from Ambion (Austin, Tex.). Both MIF and β-actin probeswere labeled with 5 μl [α-³²P]dATP (3000 Ci/mmol, 10 mCi/ml)(PerkinElmer, Boston, Mass.) using Strip-EZ™ DNA probe synthesis kit(Ambion, Austin, Tex.) and purified in ProbeQuant Microcolumns (AmershamPharmacia, Piscatany, N.J.) according to manufacturers' protocols.

RNA (10 μg) was resolved on 1.2% agarose gels at 100 volts for 1 hourand transferred to a Hybond-N+ membrane (Amersham Pharmacia, Buckingham,England) at 1.5 amps for 70 minutes on a transfer electrophoresis unit(TransPhor PowerLid, Hoefer Scientific Instruments, San Francisco). RNAwas linked to the membrane for approximately 2 minutes using a GS GeneLinker (Bio-Rad, Hercules, Calif.). The membrane was prehybridized in ahybridization oven (Sorvall Life Science, Inc., Greensboro, N.C.) inPerfect-Hyb Plus (Sigma, St. Louis, Mo.) for 1 hour at 68° C. Sheared,denatured salmon or herring testis DNA (100 μg/ml) was then added for 1hour, followed by the addition of approximately 0.1 μg probe labeled at>5×10⁸ cpm/μg. The blot was then hybridized for 12 hours at 68° C. inthe hybridization oven followed by washing at 68° C. in 2×SSC, 0.1% SDS.The membrane was washed for 1 hour, the buffer was exchanged, and thenthe membrane was washed for an additional hour at 68° C. The membranewas wrapped in Saran wrap, and mRNA was detected by Kodak X-OMAT AR Filmafter 24 hours (Eastman Kodak Co., Rochester, N.Y.). The same membranewas then re-probed in a similar manner with radiolabeled β-actin (0.1 μgprobe labeled at >5×1-08 cpm/1 g) (Ambion, Austin, Tex.). Densitometrywas performed as described above for the western blots. The β-actin mRNAbands served as a control against which to normalize the MIF mRNAdensitometry.

Ex vivo cardiac function determination by Langendorff. Mouse heartfunction was determined using the Langendorff assay procedure. Briefly,200 Units heparin sulfate were injected intraperitoneally and the micewere sacrificed 20 minutes later. The heart was immediately removed andplaced on ice in Krebs-Henseleit Buffer (2 mM NaHCO₃, 118 mM NaCl, 4.7mM KCL, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 11.1 mM glucose, pH7.4, which was prepared fresh with demineralized, deionized water andbubbled with 95% O₂ and 5% CO₂ (PO₂ 590 mmHg, pCO₂ 38 mmHg)). The aortawas cannulated with PE50 tubing, the heart perfused in a retrogrademanner through the aortic root with pre-filtered, oxygenatedKrebs-Henseleit Buffer at a constant flow rate of 1.5 ml/minute (T 37°C., 100 ml recirculating volume). The heart was placed in awater-jacketed chamber to maintain constant temperature and humidity.PE60 intramedic polyethylene tubing, connected to a Statham pressuretransducer, was inserted into the left ventricle (LV) to measure LVpressure. Temperature was monitored using a 27 gauge thermistor needleinserted into the LV muscle. After instrumentation, hearts were allowedto stabilize for 10 minutes, and hearts that failed to achieve a stablepressure or developed persistent arrhythmias during this time wereexcluded from the study. Following stabilization, LV pressure and itsfirst derivative (dP/dt), heart rate, and coronary perfusion weremeasured simultaneously with a multi-channel Grass 7D polygraph (GrassInstruments, Quincy, Mass.). Ventricular performance as a function ofcoronary perfusion was determined for all hearts by plotting peaksystolic LV pressure and ±dP/dt_(max) values against incrementalincreases in coronary flow rate.

Determination of cardiac dysfunction by echocardiography.Echocardiograms to assess systolic function were performed using M-modemeasurements. Mice were anesthestized with 5% isofluorane with 2.5 L/mO₂ for 20 seconds (until unconscience) followed by 2% isofluorane and O₂for an average of 12-15 minutes. Hair was removed from the thorax andupper abdomen using Nair® hair remover and gauze after sitting for 3minutes. Echocardiographic measurements were obtained on anesthetizedmice approximately 5-8 minutes after induction. Echocardiography wasperformed using a Hewlett-Packard Sonos 5500 (Agilent Technologies;Edmonton, Alberta, Canada) with a frame rate of 300-500 frames/second ina random and blinded manner. A 12 MHz linear transducer was placed onthe left hemithorax interfaced with a layer of US transmission gel(Aquasonic 100, Parker Laboratories; Fairfield, N.J.). The twodimensional parasternal short-axis imaging plane guided LV M-modetracings close to the papillary muscle level. Depth was set at a minimumof 2 cm with a sweep speed of 150 m/second. Tracings were printed on aSony color printer (UP-5200, Sony).

M-mode measurements. Data represent the average of nine selected cardiaccycles from at least two separate scans. End diastole was defined as themaximal LV diastolic dimension, and end systole was defined as the peakof posterior wall motion. Fractional shortening % (FS %), a surrogate ofsystolic function, was calculated from LV dimensions as follows: FS(%)=LVED−LVES/LVED×100, as shown in FIG. 12.

Statistical Analysis. Northern and Western data are expressed asmean±standard error (SE) and statistically analyzed using a OneWay-Analysis of Variance (ANOVA). A multiple comparison procedure wasemployed using the Tukey method to determine statistical significancebetween groups. Cardiac function determined by the Langendorffpreparation (including stabilization data) is expressed as the mean±SEand separate analyses were performed for each LVP, +dP/dt_(max), and−dP/dt_(max) as a function of treatment group and coronary flow rateusing a Repeated Measures-ANOVA. A multiple comparison procedureemploying the Bonferroni method was used to determine significantdifferences between groups. Serum MIF levels are expressed as themean±SE and were statistically analyzed using a One Way-ANOVA, with amultiple comparison procedure employing the Bonferroni method todetermine significance between groups. Cardiac function determined byM-mode echocardiography is expressed as fractional shortening %±SE andanalyzed using a One Way Repeated Measures-ANOVA. Additional comparisonswere performed using the Tukey Test to determine significant differencesbetween specific groups. Statistical significance for all analyses wasdefined as p≦0.05. All statistical analyses were performed usingSigmaStat 2.03 (SPSS Inc., Chicago, Ill.) and Microsoft Excel (MicrosoftCorp., Seattle, Wash.).

Results Example 13

MIF protein is constitutively expressed by cardiac myocytes in vivo andis released in response to burn injury. The cytokine macrophagemigration inhibitory factor (MIF) is present in both ventricular andatrial myocytes at baseline as demonstrated by western andimmunohistochemistry (FIGS. 7 and 8). After burn injury, a significantdecrease (2.1 fold) was identified at 8 hours with tissue concentrationsof MIF returning to baseline levels by 12 hours (FIG. 7). Thisexpression pattern was paralleled in liver, spleen, and the lung afterburn injury (FIG. 8) and is consistent with the hypothesis that MIF isreleased in response mediators of burn injury.

Systemic MIF and IL-6 levels are increased after burn injury. Maximumsystemic release of MIF (2.2 fold increase) was identified in serum at 4hours and returned to baseline levels by 8 hours (FIG. 9). Maximum serumIL-6 levels were identified at 12 hours which returned to baselinelevels by 48 hours. Serum IL-12 levels decreased after burn injury andwere minimum at 24 hours and returned to baseline by 48 hours. No othercytokines tested (as listed in the materials and methods) were detectedin the serum.

MIF mRNA in the heart significantly increases in the heart by 8 hoursafter burn injury. The levels of MIF mRNA were detected by Northernanalysis from total RNA isolated from hearts of either sham mice or miceat 4, 8, 12, 24, and 48 hours following burn injury (FIG. 10). MIF mRNAis constitutively expressed in the heart, and significant increases intranscription initially occur at 8 hours, which are upregulated for therest of the time course examined (48 hours) (FIG. 10).

Anti-MIF antibodies improve LPS-induced cardiac depression ex vivo. Theresponses of hearts to retrograde aortic perfusion at 1.5 ml/minute frommice undergoing the sham operation, burn injury, or burn injury withpre-treatment of anti-MIF antibodies were determined using a Langendorffanalysis of heart function. Significant decreases in LVP, +dP/dt_(max),−dP/dT_(max), DR, dP40, TPP, RT90 and Time to Max −dP/dt were identifiedin mice 18 hours after undergoing burn injury (TABLE 3). Micepre-treated with anti-MIF (Clone III.D.9) undergoing burn injury werecompletely protected by 18 hours (Table 3), while mice treated with theisotype control did not differ significantly from burn injury alone(data not shown).

FIG. 11 illustrates the function of hearts over a range of coronary flowrates from sham mice, burn injury mice, and burn injury mice pre-treatedwith anti-MIF antibodies 18 hours after the burn injury or shamprocedure. Increases in coronary flow resulted in incremental increasesin contractile performance in all hearts (groups) tested. Miceundergoing burn injury demonstrated a downward shift in the LVP,+dP/dt_(max), and −dP/dT_(max) function curves demonstrating significantsystolic and diastolic dysfunction (FIG. 11). This dysfunction, however,was not present when anti-MIF antibodies were given where no significantdifferences to sham mice were identified (FIG. 11).

Anti-MIF monoclonal antibody therapy improves burn injury associatedcardiac depression in vivo. Serial echocardiography was (M-mode) wasperformed on mice receiving burn injury, and mice pre-treated 90 minutesprior before burn injury with either of two anti-MIF antibodies, anisotype control, or no treatment (FIG. 12). At 4 and 8 hours, thefractional shortening percentage (FS %) of all burn injury treated micewere similarly depressed 21.4 FS (56.2 FS %-34.8 FS %), irrespective ofanti-MIF treatment. At 12 hours post burn injury, however, mice injectedwith either of the two monoclonal anti-MIF antibodies demonstratedstatistically significant recovery of FS % compared to burn injury micereceiving either no treatment of an isotype antibody control (FIG. 12).By 24 hours, the FS % of the treated mice was not significantlydifferent from the controls indicating complete protection of theassociated cardiac dysfunction. Throughout the 48 hours, the FS % ofsham mice did not change significantly indicating that the testingregimen and anesthesia did not affect cardiac function. Lastly, micereceiving isotype control antibodies did not demonstrate significantdifferences from animals undergoing burn injury alone, indicatingspecificity of the anti-MIF antibody effects. TABLE 3 In vitrostabilization data from isolated hearts in the Langendorff perfusionexperiments. Cardiac function is expressed as the mean ±SE. Separateanalyses were performed for each parameter (left column) as a functionof treatment group. A Repeated Measures ANOVA with a multiple comparisonprocedure employing the Bonferroni method was used to determinesignificant differences between groups Burn Injury + Cardiac FunctionAnti- Tested Sham (n = 11) Burn Injury (n = 9) MIF (n = 5) LVP (mmHg)96.5 ± 1.5  63.2 ± 3.3* 91.2 ± 9.6  +dP/dt max 2217 ± 44  1631 ± 40* 2256 ± 171  (mmHg/sec) −dP/dt max 1855 ± 45  1187 ± 70*  1840 ± 235 (mmHg/sec) DR 1.22 ± 0.04  1.40 ± 0.05* 1.29 ± 0.1  dP40 1868 ± 24  1383± 31*  1960 ± 150  (mmHg/sec) TPP (msec) 82.0 ± 2.5  72.8 ± 2.5* 86.2 ±2.0  RT90 (msec) 79.6 ± 4.5  68.8 ± 1.9* 84.2 ± 2.8  Time to Max 49.1 ±0.7  47.6 ± 0.6  53.0 ± 1.2  +dP/dt (msec) Time to Max 50.1 ± 0.5  45.7± 1.3* 53.6 ± 1.7  −dP/dt (msec) CPP (mmHg) 89.2 ± 4.9  91.0 ± 6.3  84.2± 5.7  CVR (mmHg) 59.4 ± 3.3  60.6 ± 4.2  56.1 ± 3.8  HR (bpm) 321 ± 6 315 ± 7  322 ± 16 (*p < 0.05 compared to sham control).

Example 14

To determine the role that TNF-α signaling has in MIF secretion and itsassociated cardiac dysfunction in a model of sublethal endotoxicosis,the following experiments were carried out in Example 14.

Materials and Methods

Antibodies and cytokines. A polyclonal rabbit anti-rat MIF IgG (TorreyPines BioLabs, Inc., Houston, Tex.) which cross reacts with murine MIFwas used for western immunoblot and immunohistochemistry. A polyclonalgoat anti-rabbit IgG-HRP (BioRad Corp., Hercules, Calif.) was used as asecondary antibody for western immunoblots. Two monoclonal mouseanti-mouse (and human) MIF IgG1 antibodies (XIV.15.5 and III.D.9, giftof Cytokine PharmaSciences, Inc., King of Prussia, Pa.) and a monoclonalmouse IgG1 isotype control antibody (HB-49, gift from CytokinePharmaSciences, Inc.) were used in the echocardiographic studies. Invivo neutralization of MIF acitivity by both the XIV.15.5 and III.D.9clones have been previously demonstrated. Recombinant human TNFR:Fc(Enbrel®) used to neutralize TNF-α was a gift from Immunex Corp./Amgen,Inc., (Thousand Oaks, Calif.). Recombinant human MIF was synthesizedaccording to the method of Bernhagen et al. and provided by CytokinePharmasciences, Inc.

Animals and Experimental Design. Pathogen-free, adult male C57BL/6J micewere obtained at 6-10 weeks of age and utilized at approximately 12weeks of age (Jackson Labs, Bar Harbor, Me.). Breeding pairs of B6:129PF1/J and B6; 129S-Tnfrsfla^(tm1 Imx)Tnfrsflb^(tm1 Imx) mice (TNFR−/−)were purchased from Jackson Laboratory (Bar Harbor, Me.) and maleoffspring were utilized after genotyping at 20-24 weeks (30-40 grams).Sterile commercial chow and water were made available ad libitum. Allanimal protocols were reviewed and approved by the University of TexasSouthwestern Medical Center Institutional Animal Care Advisory Committeeand were in compliance with the rules governing animal use as publishedby the NIH.

Mice were injected intraperitoneally with 4 mg/kg E. coli 0111:B4 LPS(Sigma-Aldrich Corp., St. Louis, Mo.) and sacrificed by CO₂ asphyxiationand subsequent cervical dislocation. Uninjected mice were used ascontrols. The two anti-MIF antibodies (III.D.9 and XIV.15.5, gift ofCytokine PharmaSciences, Inc., King of Prussia, Pa.) and their isotypecontrol (HB-49, gift from Cytokine PharmaSciences, Inc.) were injected(100 μg in 200 μg PBS) intraperitoneally 90 minutes before the LPSchallenge in the echocardiogram studies. Enbrel® (rhTNFR:Fc) wasinjected intraperitoneally (5 mg/kg or 300 μg in 0.5 ml PBS) 75 minutesprior to LPS challenge in wild type mice.

Whole hearts were removed and snap frozen in liquid nitrogen and storedat −80° C. or fixed in 10% neutral-buffered formalin for 24 hours andplaced in 70% ethanol for immunohistochemistry. Whole blood wascollected by retro-orbital bleeding and serum was separated usingseparator tubes. Serum was transferred to a sterile snap-top tube andfrozen at −80° C. until assayed by ELISA.

Determination of serum MIF levels. Sera from six mice were assayed formouse MIF using the Chemikine™ Rat/Mouse macrophage inhibitory factor(MIF) EIA kit (Chemicon International, Inc., Temecula, Calif.) accordingto the manufacturer's instructions. Briefly, 5 μl of standards, samples,or reaction buffer (blank) were added to each well in duplicate. DilutedMIF-HRP antibody conjugate was added to each well (100 μl) and allowedto incubate for 2 hours at room temperature. Wells were then washed fivetimes, and TMB substrate (100 μl) was allowed to incubate in the darkfor 30 minutes at room temperature. The stop reagent was added to eachwell, gently mixed, and the ELISA was read on an ELISA plate reader (EL312e Microplate Reader, Bio-Tek Instruments, Winooski, Vt.) at 450 nm(630 nm background) within 30 minutes of completion of the assay.

Protein Extraction and Western Blotting. Hearts stored at −80° C. werehomogenized on ice in lysate buffer (10 mM HEPES, 2 mM EDTA, 0.1% Chaps,pH 7.4) with one Complete Mini-EDTA-Free Protease inhibitor cocktailtablet per 10 ml buffer (Roche Diagnostics, Mannheim, Germany). Proteinconcentration was quantified using the Bio-Rad Protein Assay (Hercules,Calif.) and 50 μg of protein diluted in Laemmeli sample buffer (Bio-Rad)was added in a 1:1 ratio to a final volume of 10 μl and resolved on a12% SDS polyacrylamide gel under reducing conditions. PrestainedSDS-PAGE standards (Kaleidoscope Broad range, Bio-Rad Laboratories,Inc., Hercules, Calif.) were run (10 μl/lane) with each gel in order todetermine the approximate M.W. of detected bands. The gel wastransferred to a PVDF membrane (NEN, Boston, Mass.) using the MiniTransblot® electrophoretic transfer cell (Bio-Rad, Hercules, Calif.) at100 V for 70 minutes and cooled with ice packs. Subsequently, themembrane was re-wet with methanol, washed a minimum of 3 times with 100ml water, and placed in block (5% nonfat dry milk (Bio-Rad)/TBS/0.1%Tween-20 (TBS-T) overnight at 4° C. The membrane was then incubated withthe primary rabbit anti-MIF (1:1250 dilution) for 2 hours at roomtemperature in 5% milk/TBS-T. The membrane was washed 1 time for 15minutes in TBS-T, followed by five washes (5 minutes each). The membranewas then incubated for 1 hour with a HRP conjugated goat anti-rabbitantibody (diluted 1:5000) in TBS-T at room temperature. The membrane wasthen washed twice for 15 minutes, followed by five additional washes (5minutes each). The membrane was then developed for 5 minutes with 5 mlof ECL reagents (SuperSignal West Pico, Pierce, Rockford, Ill.), and theresulting chemiluminescent reaction was detected by Kodak X-OMAT AR Film(Eastman Kodak Co., Rochester, N.Y.).

Quantification of the single band density with the approximate molecularweight of MIF (12.5 kD) was determined using Quantity One software(Bio-Rad, Hercules, Calif., Ver. 4.4.0, Build 36) following conversionof radiographic film to TIFF files (8 bit grayscale) using a Scanjet3400c (Hewlett Packard, Palo Alto, Calif.) and reported in arbitraryunits (A.U.)/mm².

Immunohistochemistry. Tissue was fixed in neutral buffered formalin andprocessed to paraffin and subsequently immunostained at room temperatureon a BioTek Solutions Techmate™1000 automated immunostainer (VentanaMedical Systems, Tucson, Ariz.) using the Ultra-streptavidin biotinsystem with horseradish peroxidase and diaminobenzidine (DAB) chromogen(Signet Laboratories, Dedham, Mass.). Paraffin sections were cut at 3 μmon a rotary microtome, mounted on positively charged glass slides(POP100 capillary gap slides, Ventana Medical Systems, Tucson, Ariz.)and air-dried overnight. Sections were then deparaffinized in xylene andethanol, quenched with fresh 3% hydrogen peroxide for 10 minutes toinhibit endogenous tissue peroxidase activity, and rinsed with deionizedwater. Sections were incubated in unlabeled blocking serum for 15minutes to block nonspecific binding of the secondary antibody and thenincubated for 25 minutes with either rabbit anti-MIF (1:400, TorreyPines BioLabs, Inc., Houston, Tex.) diluted in 1% citrate buffer(BioPath, Oklahoma City, Okla.), or with buffer alone as a negativereagent control. Following washes in buffer, sections were incubated for25 minutes with a biotinylated polyvalent secondary antibody solution(containing goat anti-rabbit antibodies). Next, sections were washedwith buffer, incubated in horseradish peroxidase-conjugatedstreptavidin-biotin complex for 15 minutes, washed again in buffer, andthen incubated with 2 changes, 5 minutes each, of a freshly preparedmixture of DAB and H₂O₂ in buffer, followed by washing in buffer andthen water. Sections were then counterstained with hematoxylin,dehydrated in a graded series of ethanol and xylene, and coverslipped.Slides were reviewed by light microscopy and positive reactions with DABwere identified as a dark brown reaction product.

Total RNA isolation, MIF and β-actin probe preparation, and northernblotting. Hearts stored at −80° C. were placed in a liquid nitrogenfilled pestle and ground to a fine powder with a mortar. Each powderedheart was then immediately placed in 2 ml Trizol (Invitrogen, Carlsbad,Calif.) and total RNA was isolated according to the manufacturer'sprotocols and quantified by spectrophotometry. An MIF specific Northernprobe was prepared from an MIF containing plasmid (Image Clone I.D.634910, Research Genetics, Huntsville, Ala.) isolated using Genelute HPPlasmid MidiPrep kit (Sigma, St. Louis, MO). The fragment was preparedby an EcoR1 and Not1 digestion (Fisher Scientific, Pittsburgh, Pa.) andgel purified and isolated on a 1.2% agarose gel using GenElute AgaroseSpin Columns (Supelco, Bellefonte, Pa.). The β-actin probe DNA fragmentwas purchased from Ambion (Austin, Tex.). Both MIF and (β-actin probeswere labeled with 5 μl [α³²P]dATP (3000 Ci/mmol, 10 mCi/ml)(PerkinElmer, Boston, Mass.) using Strip-EZ™ DNA probe synthesis kit(Ambion, Austin, Tex.) and purified in ProbeQuant Microcolumns (AmershamPharmacia, Piscatany, N.J.) according to manufacturers' protocols.

Isolated total RNA (10 μg) was combined with formaldehyde loading dye(Ambion, Inc.) at a ratio of 1:3 sample:loading dye according to themanufacturer's protocols. Each gel had a 0.24-9.5 kB RNA ladder(Invitrogen Corp.) ran in parallel with samples (10 μg). Samples and RNAladder were placed at 65° C. for 10 minutes prior to electrophoresis andresolved on a 1.2% agarose gels with 1×TAE buffer (Ambion, Inc.) at 100volts for 1 hour and transferred to a Hybond-N+membrane (AmershamPharmacia, Buckingham, England) at 1.5 amps for 70 minutes on a transferelectrophoresis unit (TransPhor PowerLid, Hoefer Scientific Instruments,San Francisco) in 0.5×TAE. RNA was linked to the membrane forapproximately 2 minutes using a GS Gene Linker (Bio-Rad, Hercules,Calif.). The membrane was prehybridized in a hybridization oven (SorvallLife Science, Inc., Greensboro, N.C.) in Perfect-Hyb Plus (Sigma, St.Louis, Mo.) with sheared, denatured salmon sperm DNA (100 μg/ml) for 1hour at 68° C. The probes were prepared by heating to 90° C. for 10minutes (10 μl probe with 100 μl 10 mM EDTA), followed by the additionof approximately 0.1 μg probe labeled at >5×10⁸ cpm/μg to thehybridization buffer. The blot was then hybridized for 12 hours at 68°C. followed by washing at 68° C. in 2×SSC, 0.1% SDS. The membrane waswashed for 1 hour, the buffer was exchanged, and then the membrane waswashed for an additional hour at 68° C. The membrane was wrapped inSaran wrap, and mRNA was detected by Kodak X-OMAT AR Film after 24 hours(Eastman Kodak Co., Rochester, N.Y.). The same membrane was thenre-probed in a similar manner with radiolabeled β-actin (0.1 μg probelabeled at >5×10⁸ cpM/μg) (Ambion, Austin, Tex.). Densitometry wasperformed as described above for the western blots. The β-actin mRNAbands served as a control against which to normalize the MIF mRNAdensitometry.

Ex vivo cardiac function determination by Langendorff Mouse heartfunction was determined using the Langendorff assay procedure. Briefly,200 Units heparin sulfate were injected intraperitoneally and the micewere sacrificed 20 minutes later. The heart was immediately removed andplaced on ice in Krebs-Henseleit Buffer (2 mM NaHCO₃, 118 mM NaCl, 4.7mM KCL, 1.2 mM KH2PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 11.1 mM glucose, pH7.4, which was prepared fresh with demineralized, deionized water andbubbled with 95% O₂ and 5% CO₂(pO₂ 590 mmHg, pCO₂ 38 mmHg)). The aortawas cannulated with PE50 tubing, the heart perfused in a retrogrademanner through the aortic root with pre-filtered, oxygenatedKrebs-Henseleit Buffer at a constant flow rate of 1.5 ml/minute(constant temperature of 37° C., 100 ml recirculating volume). The heartwas placed in a water-jacketed chamber to maintain constant temperatureand humidity. Intramedic polyethylene tubing (PE60), connected to aStatham pressure transducer, was inserted into the left ventricle (LV)to measure LV pressure. Temperature was monitored using a 27 gaugethermistor needle inserted into the LV muscle. After instrumentation,hearts were allowed to stabilize for 10 minutes, and hearts that failedto achieve a stable pressure or developed persistent arrhythmias duringthis time were excluded from the study. Following stabilization, LVpressure and its first derivative (dP/dt), heart rate, and coronaryperfusion were measured simultaneously with a multi-channel Grass 7Dpolygraph (Grass Instruments, Quincy, Mass.). Ventricular performance asa function of coronary perfusion was determined for all hearts byplotting peak systolic LV pressure and ±dP/dt_(max) values againstincremental increases in coronary flow rate. Hearts were perfused withor without 20 ng/ml rMIF added to the perfusate.

Determination of cardiac dysfunction by echocardiography.Echocardiograms to assess systolic function were performed using M-modemeasurements. Mice were anesthestized with 5% isofluorane with 2.5 L/mO₂ for 20 seconds (until unconscience) followed by 2% isofluorane and O₂for an average of 12-15 minutes. Hair was removed from the thorax andupper abdomen using Nair® hair remover and gauze after sitting for 3minutes. Echocardiographic measurements were obtained on anesthetizedmice approximately 5-8 minutes after induction. Echocardiography wasperformed using an Acuson Sequoia™ Model C256 (Siemens MedicalSolutions, USA, Inc., Mountain View, Calif.) with a frame rate of300-500 frames/second in a random and blinded manner. A 15 MHz lineartransducer (15L8, Siemens Medical Solutions, USA, Inc.) was placed onthe left hemithorax interfaced with a layer of ultrasound transmissiongel (Aquasonic 100, Parker Laboratories; Fairfield, N.J.). The twodimensional parasternal short-axis imaging plane guided LV M-modetracings close to the papillary muscle level. Depth was set at a minimumof 2 cm with a sweep speed of 200 m/second.

M-mode measurements. Data represent the average of nine selected cardiaccycles from at least two separate scans. End diastole was defined as themaximal LV diastolic dimension, and end systole was defined as the peakof posterior wall motion. Fractional shortening % (FS %), a surrogate ofsystolic function, was calculated from LV dimensions as follows: FS(%)=LVED−LVES/LVED×100, as shown in FIG. 18.

Multiplex cytokine detection by Luminex. Plasma inflammatory cytokine(IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFN-γ, TNF-α, and GM-CSF)concentrations were determined using the Mouse Cytokine Ten-PlexAntibody Bead Kit (Biosource International, Inc., Camarillo, Calif.) ona Luminex xMAP™ system (Luminex Corp., Austin, Tex.) according to themanufacturer's instructions. The plate was loaded onto the Luminex XYP™platform, the instrument set to remove 50 μl, and the total event set toequal the 100 per bead set (200 collected for most). Data was collectedusing the Luminex™ Data Collector Software (Luminex Corp., Austin,Tex.). The concentrations of the lot specific reconstituted standardsused in each run were entered into the software and the analyteconcentrations for unknown samples were then extrapolated from thecytokine specific standard curve using MasterPlex™ QT software (Version1.2.8.58, Mirai Bio, Inc., Alameda, Calif.). Final concentrations weremultiplied by 2 in order to account for the initial dilution factor. Nosamples were detected that were higher than the standards curves for anycytokine.

Statistical Analysis. Northern and Western data are expressed asmean±standard error (SE) and statistically analyzed using a OneWay-Analysis of Variance (ANOVA). A multiple comparison procedure wasemployed using the Tukey method to determine statistical significancebetween groups. Cardiac function determined by the Langendorffpreparation (including stabilization data) is expressed as the mean±SEand separate analyses were performed for each LVP, +dP/dt_(max), and−dP/dt_(max) as a function of treatment group and coronary flow rateusing a Repeated Measures-ANOVA. A multiple comparison procedureemploying the Bonferroni method was used to determine significantdifferences between groups. Serum MIF levels are expressed as themean±SE and were statistically analyzed using a One Way-ANOVA, with amultiple comparison procedure employing the Bonferroni method todetermine significance between groups. Cardiac function determined byM-mode echocardiography is expressed as fractional shortening %±SE andanalyzed using a One Way Repeated Measures-ANOVA. Additional comparisonswere performed using the Tukey Test to determine significant differencesbetween specific groups. Statistical significance for all analyses wasdefined as p<0.05. All statistical analyses were performed usingSigmaStat 2.03 (SPSS Inc., Chicago, Ill.) and Microsoft Excel (MicrosoftCorp., Seattle, Wash.).

Results Example 14

Serum MIF levels in WT mice, WT mice pre-treated with Enbrel®, andTNFR−/− mice. Serum levels of MIF reach maximum (˜1.5 fold baseline) at8 hours in wild type mice after LPS challenge (FIG. 13A). When TNFR−/−mice are challenged with LPS, maximum serum MIF levels occur as 12 hours(˜1.7 fold baseline)(FIG. 13B). Maximum serum MIF levels (˜2.3 foldbaseline) were identified in wild type mice pre-treated (60 minutes)with Enbrel® and challenged with LPS at 24 hours (FIG. 13C).

Cardiac MIF is not released from the heart, spleen, or liver in TNFR−/−mice after LPS challenge. Both western and immunohistochemistry analysisperformed on cardiac, liver, and spleen demonstrated that previouslydocumented release in wild type mice after LPS challenge did not occurat any time in TNFR−/− mice or wild type mice pre-treated with Enbrel®,both of which prevent TNF-α signaling (FIGS. 14 and 15).

Cardiac MIF transcription is not modulated in TNFR−/− mice after LPSchallenge. Detection of MIF mRNA from isolated total RNA from hearttissue from TNFR−/− mice challenge with LPS demonstrates thattranscription of MIF is not upregulated after LPS challenge (FIG. 16),which has been identified in wild type mice previously at 48 hours.

MIF has direct cardiodepressant effects in TNFR−/− mice to the sameextent as in WT mice. To determine if MIF directly influences cardiacfunction independently of TNF, spontaneously beating normal mouse hearts(Langendorff preparation) were perfused with recombinant human MIF(rMIF) at a concentration of 20 ng/ml, approximating the documentedserum levels in patients with septic shock (25). The human MIF used inthe Langendorff perfusion studies has been shown to have anapproximately 90% homology with murine MIF and has been shown to havecross species biologic function.

Table 4 demonstrates that the responses of the background strain ofTNFR−/− mice, C57BL/6 mice, and the TNFR−/− mice to retrograde aorticperfusion at 1.5 ml/minute with control perfusate or perfusatecontaining 20 ng/ml rMIF. Perfusion with rMIF led to a significantdecrease in LVP, +dP/dt_(max), −dP/dT_(max), and dp40 (mm Hg/sec) inboth mouse strains while other parameters (time to max ±dP/dt, CPP, CVR,and HR) were unaffected. FIG. 18 illustrates the effect of rMIF over arange of coronary flow rates. Increases in coronary resulted in astep-wise increase in contractile performance regardless of experimentalgroup assignment. MIF challenged hearts demonstrated a downward shift infunction curves, resulting in significant systolic (+dP/dt) anddiastolic (−dP/dt) function curves in response to 20 ng/ml (p<0.05).

MIF neutralization by anti MIF antibodies results in complete protectionat 24-48 hours after LPS challenge in TNFR−/− mice. To determine theinfluence of TNF signaling on MIF in the pathogenesis of cardiacdysfunction in vivo, serial echocardiography (M-mode) was performed onLPS challenged TNFR−/− mice pretreated (90 minutes prior) with either oftwo anti-MIF monoclonal antibodies, an isotype control antibody, or notreatment (FIG. 17). At 4, 8, and 12 hours after LPS challenge, thefractional shortening percentage (FS %) of all LPS challenged mice wassimilarly depressed (27.7+/−0.01 FS %) compared to baseline(45.9+/−0.002 FS %), irrespective of group assignment (FIGS. 17B, C, E).At 24 hours after LPS challenge, however, mice treated with either ofthe two monoclonal anti-MIF antibodies demonstrated statisticallysignificant recovery of FS % compared to LPS challenged group receive orLPS or LPS and the isotype antibody (FIG. 17). This enhanced recovery offunction persisted at 48 hours where function was completely restoredand LPS challenged mice receiving the isotype control were stillprofoundly depressed (FIGS. 17D, 17E). Throughout the 48 hours, the FS %of untreated control TNFR−/− mice did not change significantlyindicating that cardiac function was unaffected the testing regimen.

Serum cytokine release in wild type mice and TNFR−/− mice. Since otherinflammatory cytokines have been shown to play a role in cardiacdysfunction in addition to TNF-α early after LPS challenge (i.e. IL-1β,IL-6), we determined serum levels of an inflammatory panel in wild typeand TNFR−/− mice (FIG. 19). Not obvious in this figure are the releaseof TNF-α, IL-1β in wild type mice because of the significant increasesin these cytokines in TNFR−/− mice (31.2 (4934/158 pg/ml) fold and 94.7(7099/75 pg/ml) fold increase over wild type at 4 hours after LPSchallenge) as shown in FIGS. 19A and 19B. Similarly, and IL-12 wasincreased in the TNFR−/− mice compared to wild type mice (and 1.7(5128/2937 pg/ml) fold) (FIG. 19C), while IFN-γ levels were decreased3.6 (210/58 pg/ml) fold (FIG. 19D). IL-10 and IL-6 increased similarlyin wild type and TNFR−/− mice, although the delay of each of thesecytokines was diminished in the TNFR−/− mice (FIGS. 19E and 19F). IL-6levels were 8.5 fold in the TNFRKO mice at 4 hours compared to wild typemice (7099/835 pg/ml). Systemic increases in GM-CSF were identified inboth wild type and TNFR−/− mice and the temporal response was nearlyidentical (FIG. 19G). The cytokines IL-2, IL-4, and IL-5 demonstrated nonegligible modulation after LPS challenge in either wild type or TNFR−/−mice.

Serum cytokine release after MIF neutralization in wild type mice. Ofthe 10 cytokines assayed for in this study, MIF neutralizing antibodies(pre-LPS challenge) only affected the modulation (increase or decrease)of serum IFN-γ and IL-10 levels after LPS challenge in wild type mice(FIG. 20). Specifically, the release of IFN-γ peak at 8 hours after LPSchallenge (FIG. 19C) was attenuated 3 fold (210/69 pg/ml)(FIG. 20A). Thedelayed release of IL-10 in wild type mice which peaked at 48 hours wasattenuated 2.9 fold (244/84 pg/ml)(FIG. 20B) after LPS challenge in wildtype mice. TABLE 4 In vitro stabilization data from isolated hearts inthe Langendorff perfusion experiments. Cardiac function is expressed asthe mean ± SE. Separate analyses were performed for each parameter (leftcolumn) as a function of treatment group and coronary flow rate. ARepeated Measures ANOVA with a multiple comparison procedure employingthe Bonferroni method was used to determine significant differencesbetween groups (*p < 0.05 compared to control). Wild Type TNF KO + KrebsTNF KO + rMIF WT + rMIF TNF KO (n = 8) (WT) (n = 6) P (n = 4) p (n = 4)P (n = 5) P LVP 101.5 ± 4.3  103.3 ± 3.1  NS 102.5 ± 2.5  NS 66.0 ± 4.70.005 55.6 ± 5.9 0.001 (mmHg) +dP/dt max 2488 ± 74  2280 ± 70  NS 2475 ±48  NS 1800 ± 100 0.002 1400 ± 158 0.001 (mmHg/sec) −dP/dt max 2016 ±59  1976 ± 87  NS 2175 ± 63  NS 1413 ± 151 0.03  1070 ± 146 0.005(mmHg/sec) DR  1.24 ± 0.05  1.16 ± 0.05 NS  1.14 ± 0.02 NS  1.30 ± 0.10NS  1.32 ± 0.06 NS dP40(mmHg/sec) 2106 ± 63  1940 ± 51  NS 2175 ± 94  NS1613 ± 105 0.006 1240 ± 174 0.005 TPP (msec) 70.4 ± 2.1 64.2 ± 4.1 NS71.5 ± 2.5 NS 67.0 ± 2.4 NS 71.6 ± 1.6 NS RT90 (msec) 71.1 ± 2.1 72.2 ±4.6 NS 68.0 ± 3.0 NS 71.3 ± 4.2 NS 75.2 ± 2.9 NS Time to Max +dP/dt 49.3± 0.4 44.6 ± 2.0 NS 48.3 ± 1.2 NS 47.0 ± 2.4 NS 49.2 ± 0.5 NS (msec)Time to Max 50.0 ± 1.0 49.4 ± 0.8 NS 50.0 ± 3.5 NS 47.0 ± 2.4 NS 52.4 ±1.7 NS −dP/dt (msec) CPP (mmHg) 89.3 ± 5.7 86.4 ± 5.3 NS 70.0 ± 5.3 NS91.5 ± 6.6 NS 84.8 ± 8.6 NS CVR (mmHg) 59.5 ± 3.8 57.6 ± 3.5 NS 46.7 ±3.5 NS 61.0 ± 4.4 NS 56.5 ± 5.7 NS HR (bpm) 330 ± 14 362 ± 16 NS 318 ±21 NS 335 ± 20 NS 338 ± 10 NS

Example 15

Animals: C57BL/6 mice from Charles River (12-15 weeks old) weremaintained on commercial chow and tap water ad libitum. All animalprotocols were reviewed and approved by the University of TexasSouthwestern IACAC in compliance with the rules governing animal usepublished by NIH.

Coronary artery ligation: Mice were anesthetized with 1-1.5% isofluraneafter which coronary artery ligation was performed. Atropine (0.75 mg/kggiven intramuscularly), lidocaine (1 mg/kg intramuscularly), and saline(1 ml intraperitoneally) were given pre-operatively. Ventilation wasachieved using a custom mask fitted to the mouse snout and a smallanimal Ventilator (Harvard Apparatus, Inc., Holliston, Mass.). Anincision (˜5 mm) was made in the left thorax in the fourth intercostalspace and pericardiotomy was performed to expose the left ventricle. Theleft coronary artery was occluded using 8-0 prolene approximately 2 mmunder the left auricle. Subsequently, the chest was closed in layers andthe negative pressure of the chest returned by syringe evacuation.Buprenorphine (0.10 mg/kg) was given once post-operatively for pain.Sham procedures were performed identically without the coronaryligation.

Anti-MIF antibody: A monoclonal anti-mouse MIF IgG1 antibodies (III.D.9,gift from Cytokine PharmaScience, Inc.) and a monoclonal IgG1 isotypecontrol antibody (HB-49, gift from Cytokine PharmaScience, Inc.) wereused in the echocardiographic studies. Previous studies havedemonstrated in vivo neutralization of MIF activity. FIGS. 21-25 showthe results obtained in this example. FIG. 21 Compares cardiac function(fractional shortening) in post LAD ligation with LAD only andanti-MIF+LAD. FIG. 22 Shows the effect of anti-MIF therapy pre-LAD withLAD only and anti-MIF+LAD. FIG. 23 Presents cardiac function data 48hours post-LAD for several treatment groups. FIG. 24 Shows the serumtroponin concentration 48 hrs post-LAD with pre- and delayed anti-MIFtreatment. FIG. 25 Shows the serum troponin I and MIF concentrationsthrough two weeks post ligation.

Example 16

MIF is secreted from cardiomyocytes following LPS challenge, anddirectly mediates a late onset (>6 hours) cardiac dysfunction. In immunecells, CD74 was recently determined to be the MIF receptor, exertingeffects via ERK1/2 intracellular signaling pathways. To determine ifCD74 mediates MIF-induced cardiac dysfunction in sepsis, wechallenged: 1) wild type mice (C57BL/6) with LPS; 2) wild type micepre-treated with anti-CD74 monocolonal neutralizing antibodies; andchallenged with LPS, and 3) CD74 knock-out mice with LPS (4 mg/kg).Serial echocardiography was performed and fractional shortening (FS %)was determined. At 24 hours, significant dysfunction was observed in WTmice given LPS (FS %=31.6%±3.3%) compare to controls (FS %=58±1%). Inboth anti-CD74 antibody treated and CD74 knock-out mice challenged withLPS, cardiac function was significantly improved compared to wild typemice given LPS alone (FS %=49±3.6% and 53.3±2.4%, respectively, p<0.05).As CD74 expression has never been documented in the heart, we performedimmunoblots and histochemistry which confirmed that CD74 wasconstituitively present on cardiac cell membranes and in the cytosol;and was substantially regulated after LPS challenge (nearly absent at 12hours->4 fold decrease). FIGS. 26-30 and Tables 5-7. These data are thefirst to demonstrate that CD74 is expressed on cardiomyocytes and is acritical mediator of cardiac dysfunction. TABLE 5 Gel name: CD74Series 1(Raw 1-D Image) Area Mean Value Density Index mm2 INT Std. DeviationINT/mm2 1 60.752571 0.194609751 0.189491850143 27.14811295 2 60.7525710.287409566 0.248436881024 40.09371219 3 60.752571 0.2339610150.202269192827 32.63762488 4 60.752571 0.193959627 0.18027135095527.05742043 5 60.752571 0.395414888 0.260615830635 55.16048369 660.752571 0.324963155 0.260421026338 45.33244800 7 60.752571 0.3837205150.267718631786 53.52911561 8 60.752571 0.445625310 0.25655673818262.16485123 9 60.752571 0.424732026 0.248033724362 59.25023237 1060.752571 0.391370235 0243462155963 54.59625355 11 60.752571 0.6072513150.202530867340 84.71172258 12 60.752571 0.620208919 0.18495139971786.51931171Background Subtraction Method: LocalData units: Intensity (INT)

TABLE 6 CD74 KO MOUSE - rh/MIF added to perfusate p CD74 p CD74 CD74 KOKO + rhMIF C57BL/6 CD74 C57BL/6 + rhMIF KO (n = 3) (n = 3) p (n = 3) KO(n = 3) p rhMIF Left Ventricular Pressure (mmHg) 104.7 ± 12.9  67.3 ±14.4 NS 96.0 ± 3.0 NS 52.7 ± 6.6 0.004 NS +dP/dt max (mmHg/sec) 22533 ±260  1867 ± 176 NS 2333 ± 33  NS 1417 ± 164 0.005 NS −dP/dt max(mmHg/sec) 2250 ± 150 1433 ± 202 0.03 2033 ± 33  NS 1233 ± 159 0.008 NSDifferential Ratio  1.12 ± 0.06  1.32 ± 0.18 NS  1.15 ± 0.05 NS  1.15 ±0.02 NS NS dP40 (mmHg/sec) 2200 ± 115 1662 ± 120 0.03 1867 ± 67  NS 1200± 115 0.007 0.05 Time to Peak Pressue (msec) 77.3 ± 6.3 69.3 ± 3.5 NS78.0 ± 4.1 NS 74.0 ± 3.1 NS NS Time to 90% Relaxation (msec) 73.3 ± 3.377.3 ± 2.7 NS 81.0 ± 5.9 NS 73.3 ± 4.4 NS NS Time to Max +dP/dt (msec)51.0 ± 2.1 45.0 ± 2.9 NS 51.0 ± 2.1 NS 47.0 ± 2.1 NS NS Time to Max−dP/dt (msec) 49.7 ± 3.3 52.7 ± 0.3 NS 51.7 ± 1.7 NS 49.0 ± 1.5 NS NSCoronay Perfusion Pressue (mmHg)  80.0 ± 15.5  86.0 ± 23.6 NS  80.0 ±20.0 NS 86.0 ± 23  NS NS Coronay Vascular Resistance 56.3 ± 7.6 56.3 ±7.7 NS  53.3 ± 13.3 NS 57.3 ± 7.7 NS NS (mmHg) Heart Rate (bpm) 313 ± 2 322 ± 2  NS 325 ± 8  NS 318 ± 2  NS NS

TABLE 7 CD74 K/O LVP +dP/dt −dP/dt DR dP40 TPP 10/24/03 124 3000 24001.25 2400 90 11/4/03.1 80 2100 1950 1.076923 2000 72 11/4/03.2 110 25002400 1.041667 2200 70 n 3 3 3 3 3 3 x 104.6666667 2533.333 2250 1.1228632200 77.33333 sd 22.4796204 450.925 259.8076 0.111506 200 11.01514 se12.97861489 260.3417 150 0.064378 115.4701 6.359595 CCD74 K/O RT90 Max +d Max − d CPP CVR HR 10/24/03 80 55 56 102 45 250 11/4/03.1 70 48 45 5053 370 11/4/03.2 70 50 48 88 71 320 n 3 3 3 3 3 3 x 73.33333 51 49.6666780 56.33333 313.3333 sd 5.773503 3.605551 5.686241 26.90725 13.3166660.27714 se 3.333333 2.081666 3.282953 15.53491 7.688375 34.80102 CD74K/O + rhMIF LVP +dP/dt −dP/dt DR dP40 TPP 10/24/03 64 1800 1400 1.2857141600 70 11/4/03.1 50 1600 1100 1.454545 1500 75 11/4/03.2 88 2200 18001.222222 1900 63 n 3 3 3 3 3 3 x 67.33333333 1866.667 1433.333 1.3208271666.667 69.33333 sd 19.21804707 305.505 351.1885 0.120076 208.16666.027714 se 11.09554465 176.3834 202.7588 0.069326 120.185 3.480102 p0.094066011 0.101343 0.031734 0.104538 0.032901 0.331722 CD74 K/O RT90Max + d Max − d CPP CVR HR 10/24/03 80 45 53 118 45 320 11/4/03.1 80 5052 40 53 320 11/4/03.2 72 40 53 100 71 325 n 3 3 3 3 3 3 x 77.33333 4552.66667 86 56.33333 321.6667 sd 4.618802 5 0.57735 40.84116 13.316662.886751 se 2.666667 2.886751 0.333333 23.57965 7.688375 1.666667 p0.401788 0.167103 0.414702 0.842117 1 0.82272 C57BL/6 LVP +dP/dt −dP/dtDR dP40 TPP 10/27/03.1 90 2300 2000 1.15 2000 84 10/27/03.2 98 2400 20001.2 1800 80 10/27/03.3 100 2300 2100 1.095238 1800 70 n 3 3 3 3 3 3 x 962333.333 2033.333 1.148413 1866.667 78 sd 5.291502622 57.73503 57.735030.052399 115.4701 7.211103 se 3.055050463 33.33333 33.33333 0.03025366.666667 4.163332 pCD74 0.551135037 0.488514 0.231336 0.737615 0.0667670.934326 C57BL/6 RT90 Max + d Max − d CPP CVR HR 10/27/03.1 83 50 50 10066.66667 315 10/27/03.2 90 55 55 100 66.66667 320 10/27/03.3 70 48 50 4026.66667 340 n 3 3 3 3 3 3 x 81 51 51.66667 80 53.33333 325 sd 10.148893.605551 2.886571 34.64102 23.09401 13.22876 se 5.859465 2.0816661.666667 20 13.33333 7.637626 pCD74 0.318926 1 0.615837 1 0.8549580.759751 C57BL/6 + rhMIF LVP +dP/dt −dP/dt DR dP40 TPP 10/27/03.1 621650 1500 1.1 1400 72 10/27/03.2 40 1100 950 1.157895 1000 80 10/27/03.356 1500 1250 1.2 1200 70 n 3 3 3 3 3 3 x 52.66666667 1416.667 1233.3331.152632 1200 74 sd 11.37248141 284.312 275.3785 0.050207 200 5.291503se 6.565905201 164.1476 158.9899 0.028987 115.4701 3.05505 p 0.0039213490.005425 0.007903 0.924639 0.00749 0.481817 p.CD74MIE 0.3188084350.135201 0.480962 0.088782 0.048812 0.37059 C57BL/6 + rhMIF RT90 Max + dMax − d CPP CVR HR 10/27/03.1 80 48 52 108 72 320 10/27/03.2 75 50 48110 73.33333 320 10/27/03.3 65 43 47 40 26.66667 315 n 3 3 3 3 3 3 x73.33333 47 49 86 57.33333 318.3333 sd 7.637626 3.605551 2.64575139.84972 26.56648 2.886751 se 4.409586 2.081666 1.527525 23.0072515.33816 1.666667 p 0.354827 0.245802 0.303548 0.853566 0.8535660.441823 p.CD74MIE 0.480962 0.604145 0.078928 1 0.956318 0.2302

Example 17

Coronary artery ligation. Mice were anesthetized with 1-1.5% isofluraneafter which coronary artery ligation was performed. Atropine (0.075mg/kg given intramuscularly), lidocaine (1 mg/kg intramuscularly), andsaline (1 ml intraperitoneally) were given pre-operatively. Ventilationwas achieved using a custom mask fitted to the mouse snout and a smallanimal ventilator (Harvard Apparatus, Inc., Holliston, Mass.). Anincision (˜5 mm) was made in the left thorax in the fourth intercostalspace and pericardiotomy was performed to expose the left ventricle. Theleft coronary artery was occluded using 8-0 prolene approximately 2 mmunder the left auricle. Subsequently, the chest was closed in layers andthe negative pressure of the chest returned by syringe evacuation. Anthe acid methyl ester of(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic (“ISO-1”,“CPSI-26” or p-hydroxyphenol-isoxazoline methyl ester) was given at adosage of 200 mg/kg in 25 microliters DMSO intraperitoneally daily fortwo weeks. Buprenorphine (0.10 mg/kg) was given once post-operativelyfor pain. Sham procedures were performed identically without thecoronary ligation. The results are shown in FIG. 31. Abbreviations: ABCtransporters, ATP binding cassette transporters; FS %, fractionalshortening %; IL-10, interleukin-1beta; IRAK, IL-1 receptor-associatedkinase-M; LPS, lipopolysaccharide; MDA, malondialdehyde; MIF,(macrophage) migration inhibitory factor; Tlr-4, toll-like receptor-4;CLP, cecal ligation and puncture; Enbrel®, trade name for recombinanthuman TNFR:Fc (soluble TNF receptor that neutralizes TNF activity invivo); IL-1β, interleukin-1beta; TNF-α, tumor necrosis factor-alpha;TNFR−/−, TNF-α receptor I/receptor II knock-out mice.

The entirety of each of the references cited herein, above and below, isincorporated herein by reference for all purposes.

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Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically recited herein.

1. A pharmaceutical composition effective for at least one selected fromthe group consisting of treating or preventing cardiac dysfunction in asubject in need thereof, treating or preventing irregularity inmyocardial activity in a subject in need thereof, treating or preventingdepression in myocardial activity in a subject in need thereof, treatingor preventing burn-injury associated cardiac dysfunction in a subject inneed thereof, treating or preventing cardiac dysfunction following acutemyocardial infarction in a subject in need thereof, treating orpreventing cardiodepression in a subject in need thereof, and acombination thereof, comprising: an effective amount of at least oneanti-MIF antibody; and at least one pharmaceutically acceptable carrier.2. The pharmaceutical composition of claim 1, further comprising atleast one CD74 inhibitor.
 3. The pharmaceutical composition of claim 2,wherein said CD74 inhibitor comprises at least one anti-CD74 antibody.4. A pharmaceutical composition effective for at least one selected fromthe group consisting of treating or preventing cardiac dysfunction in asubject in need thereof, treating or preventing irregularity inmyocardial activity in a subject in need thereof, treating or preventingdepression in myocardial activity in a subject in need thereof, treatingor preventing burn-injury associated cardiac dysfunction in a subject inneed thereof, treating or preventing cardiac dysfunction following acutemyocardial infarction in a subject in need thereof, treating orpreventing cardiodepression in a subject in need thereof, and acombination thereof, comprising: an effective amount of at least oneanti-CD74 antibody, and at least one pharmaceutically acceptablecarrier.
 5. A pharmaceutical composition effective for at least oneselected from the group consisting of treating or preventing cardiacdysfunction in a subject in need thereof, treating or preventingirregularity in myocardial activity in a subject in need thereof,treating or preventing depression in myocardial activity in a subject inneed thereof, treating or preventing burn-injury associated cardiacdysfunction in a subject in need thereof, treating or preventing cardiacdysfunction following acute myocardial infarction in a subject in needthereof, treating or preventing cardiodepression in a subject in needthereof, and a combination thereof, comprising: an effective amount ofat least one anti-TNFR antibody; an effective amount of at least oneanti-MIF antibody; and at least one pharmaceutically acceptable carrier.6. A method for treating or preventing cardiac dysfunction in a subject,said method comprising: administering to said subject an effectiveamount of at least one anti-MIF antibody.
 7. The method of claim 6,wherein said cardiac dysfunction is selected from the group consistingof irregularity in myocardial activity, depression in myocardialactivity, and a combination thereof.
 8. A method for treating orpreventing burn injury-associated cardiac dysfunction in a subject, saidmethod comprising: administering to said subject an effective amount ofat least one anti-MIF antibody.
 9. The method of claim 8, wherein saidburn injury-associated cardiac dysfunction is selected from the groupconsisting of irregularity in myocardial activity, depression inmyocardial activity, and a combination thereof.
 10. A method fortreating or preventing cardiac dysfunction in a subject, said methodcomprising: administering to said subject an effective amount of atleast one anti-CD74 antibody.
 11. A method for improving cardiacfunction in a subject following acute myocardial infarction, said methodcomprising: administering to said subject an effective amount of atleast one anti-MIF antibody.
 12. A method for at least one selected fromthe group consisting of treating or preventing cardiac dysfunction in asubject in need thereof, treating or preventing irregularity inmyocardial activity in a subject in need thereof, treating or preventingdepression in myocardial activity in a subject in need thereof, treatingor preventing burn-injury associated cardiac dysfunction in a subject inneed thereof, treating or preventing cardiac dysfunction following acutemyocardial infarction in a subject in need thereof, treating orpreventing cardiodepression in a subject in need thereof, and acombination thereof, comprising administering to a subject in needthereof an effective amount of at least one anti-TNFR antibody; andoptionally, at least one pharmaceutically acceptable carrier.
 13. Amethod for identifying an MIF inhibitor, said method comprising:exposing at least one myocyte to at least one MIF; determining at leastone MIF-related myocyte activity; exposing said myocyte to said MIF andat least one candidate agent; determining said MIF-related myocyteactivity in the presence of said candidate agent; and determiningwhether said candidate agent affects said MIF-related myocyte activity.14. A method for treating or preventing or preventing cardiacdysfunction in a subject following acute myocardial infarction, saidmethod comprising: administering to said subject an effective amount ofat least one anti-TNFR antibody and an effective amount of at least oneanti-MIF antibody.
 15. A method for treating or preventing cardiacdysfunction in a subject, said method comprising: administering to saidsubject an effective amount of at least one anti-MIF antibody.
 16. Amethod for treating or preventing burn injury-associated cardiacdysfunction in a subject, said method comprising: administering to saidsubject an effective amount of a composition comprising at least oneanti-MIF antibody and at least one pharmaceutically acceptable carrier.17. A method for treating or preventing cardiac dysfunction in asubject, said method comprising: administering to said subject aneffective amount of a composition comprising at least one anti-CD74antibody and at least one pharmaceutically acceptable carrier.
 18. Amethod for improving cardiac function in a subject following acutemyocardial infarction, said method comprising: administering to saidsubject an effective amount of a composition comprising at least oneanti-MIF antibody and at least one pharmaceutically acceptable carrier.19. A method for treating or preventing cardiac dysfunction in a subjectfollowing acute myocardial infarction, said method comprising:administering to said subject an effective amount of a compositioncomprising at least one anti-TNFR antibody, at least one anti-MIFantibody, and at least one pharmaceutically acceptable carrier.
 20. Amethod for at least one selected from the group consisting of treatingor preventing cardiac dysfunction in a subject in need thereof, treatingor preventing irregularity in myocardial activity in a subject in needthereof, treating or preventing depression in myocardial activity in asubject in need thereof, treating or preventing burn-injury associatedcardiac dysfunction in a subject in need thereof, treating or preventingcardiac dysfunction following acute myocardial infarction in a subjectin need thereof, treating or preventing cardiodepression in a subject inneed thereof, and a combination thereof, comprising administering tosaid subject an effective amount of at least one selected from the groupconsisting of small molecule MIF inhibitor, salt thereof, prodrugthereof, and a combination thereof.